BIO 241 CELL BIOLOGY LAB MANUAL

INSTRUCTOR: Nesrin zren LAB ASSISTANTS: Sibel Uur

Tolga Aslan Tuncay eker idem Atay

Boazii University Department of Molecular Biology and Genetics 2007-2008 FALL

1 BOAZII UNIVERSITY DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS 2007-2008 FALL TABLE OF CONTENTS
Grading of the Course..................................................................................................................... 2 A PREVIEW: THE WORLD OF THE CELL GUIDELINES .................................................... 3 How to Keep a Lab Notebook ........................................................................................................ 4 How to Write a Lab Report .............................................................................................................. 5 Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 Experiment 8 Experiment 9 : Microscopic Measurements.............................................................................. 10 : Plasma Membrane ............................................................................................ 14 : Cellular Fractionation ....................................................................................... 17 : Analysis of Subcellular Fractions I .................................................................. 21 : Analysis of Subcellular Fractions II ................................................................. 26 : DNA Extraction From Bovine Spleen.............................................................. 29 : Mitosis and Cytokinesis ................................................................................... 32 : Cell Culture ...................................................................................................... 35 : Analysis of Polysaccharides ............................................................................. 39

Experiment 10 : Cellular Carbohydrates ..................................................................................... 43

TT 7-8 Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 Experiment 8 Experiment 9 Experiment 10 Microscopic Measurements Plasma Membrane Cellular Fractionation Analysis of Subcellular Fractions I Analysis of Subcellular Fractions II DNA Extraction From Bovine Spleen Mitosis and Cytokinesis Cell Culture Analysis of Polysaccharides Cellular Carbohydrates Review Lab Final

ThTh

2-Oct-07 4-Oct-07 9-Oct-07 11-Oct-07 16-Oct-07 18-Oct-07 23-Oct-07 25-Oct-07 30-Oct-07 1-Nov-07 6-Nov-07 8-Nov-07 13-Nov-07 15-Nov-07 20-Nov-07 22-Nov-07 27-Nov-07 29-Nov-07 4-Dec-07 6-Dec-07 11-Dec-07 13-Dec-07 With course final

GRADING OF THE COURSE

Your lab grade makes up 20% of your course grade and it should be at least 45/100 in order to be successful in Bio241. Lab Reports accepted after 3 days) Introduction Purpose Materials Procedure Results-Discussion-Questions References 1.5/10 1.0/10 0.5/10 1.0/10 5.0/10 1.0/10 10/20

(submission at the beginning of following lab session. 1 for each day late and not

(Lab manual as a reference is not accepted) Quiz 3/20

Lab Final

7/20

Questions: please contact bio241lab@yahoo.com.tr

THE WORLD OF THE CELL: A PREVIEW

The cell is the basic unit of life. Every organism either consists of cells or is itself a single cell. Only as we understand the structure and function of cells can we appreciate both the capabilities and limitations of living organisms, whether animal, plant or microorganism. We are in the midst of a revolution in biology that has brought with it tremendous advances in our understanding of how cells carry out the intricate functions necessary for life. Particularly significant is the dynamic nature of a cell, as evidenced by its capacity to grow, reproduce and become specialized and by its ability to respond to stimuli and to adapt to changes in its environment. Cell biology itself is changing, as scientists from a variety of related disciplines focus their efforts on the common objective of understanding how cells work. The convergence of cytology, molecular genetics and biochemistry has made modern cell biology one of the most exciting and dynamic disciplines in contemporary biology.

How to Keep a Lab Notebook

Guide for Students and Laboratory Instructors Department of Molecular Biology and Genetics

The most important aspect of scientific investigation is keeping track of your work. There is no point in performing work that will certainly be lost, as time efficiently erases memory, people, and even single copies of anything. In a world of increasing standards, GLP (good laboratory practice) is in itself becoming an area of expertise. A good lab notebook should contain sufficient detail for any other person to be able to repeat the same experiment and achieve the same results, reproducibility being a primordial requisite for a phenomenon to become a scientific fact. Legally, all proprietary rights conflicts are resolved primarily by investigation of lab notebooks. In the case of industrial labs, notebooks belong to the laboratory and must include details such as make and lot number of reagents, date of preparation of solutions, names of persons that prepared them. Each page of the notebook is numbered, dated, signed by investigator and countersigned by controller. In student labs, we need not go that far; the minimum requirements are given below. LABORATORY NOTEBOOK: Use a single, bound-page notebook, never use loose notepaper. Always include experiment date and title. Make notes as you go along. Note everything you use and do, keeping in mind the rule that someone else should be able to repeat what you did by reading your notes. You may refer to published protocols/methods, but must note any modifications or specific conditions. Note details of calculations, such as for solutions or dilution series, including volumes actually used. Paste all figures, photos, printouts etc. in notebook. Every observation is important.

How to Write a Lab Report

Preparation and Evaluation Guide for Students and Laboratory Instructors Department of Molecular Biology and Genetics

This guide provides a standard format in which all lab reports, in all courses, must be written. The goal of the lab report, like a scientific paper, is to convey to your audience why the experiment was done (background) what was done (materials and methods), what was the outcome (results), the significance of the results (discussion), and the published sources that assisted your experimentation or interpretation (references). Some courses may require slight alterations or changes to this format, and these will be explained at the beginning of the lab. The basis for writing a good lab report, like writing a research paper, is an organized record of the experiments and your results. A lab notebook is the place where experiments are described in great detail, and the raw data is recorded.

LAB REPORT REQUIREMENTS 1. Keeping a detailed lab notebook is a prerequisite. Please see the attached Lab Notebook Guidelines. 2. Reports must be original work. Although experiments may be carried out as a group, each report must be written individually. Plagiarism will result in a grade of zero for the report. 3. Reports must be type written and subjected to a spell check. Grammar and spelling mistakes are the first indication of sloppy work.

6 Lab Report Format Each section must be included in each report, unless told differently by the lab instructor.

I. Title page A. Experiment name B. Experimenter (author) and partners (co-authors) C. Experiment date(s) D. Evaluator E. Submission date

II. Introduction A. Provides a brief summary of the background and theory pertaining to the experiment done. B. Material for the introduction can be found in books, articles, your lab manual or the internet (but only from reliable sites be careful!). Any information that is not general knowledge must be referenced appropriately. Plagiarism is easily recognized. C. Must answer the questions of; What was known before the experiment was done? Why was the experiment carried out? Was a hypothesis being tested? If so, the hypothesis must be specifically stated.

III. Purpose Few sentences in order to answer; why was this experiment performed?

IV. Materials and Methods A. What materials and reagents were used? B. What was done step by step in your own words (not copied directly from lab manual)? Diagrams and flow charts are welcome. C. Alterations, mistakes or corrections must be included. Very important!!!

7 V. Results A. Informing your audience of the purpose of the experiment In order to measure the pH of X or To ask whether the sample contained enzyme X B. State the direct outcome of the experiment or procedure. note the difference between what you see, what you think you see, and what you think it means (Moriarty, 1997) C. Each experiment should answer a simple question i.e. what do the cells of an algae look like? or what is the concentration of cells in an unknown sample?). Each answer should be represented by the results of your experiment both in table or figure AND in words. When writing a description, your audience should be able to use your words to reconstruct what you observed. D. The following questions, as suggested by Moriarty (pgs. 89-90) (Moriarty, 1997), should be answered in the result section of any piece of scientific writing Why did you conduct the experiment? What did you do? What did you see? What does it mean? This point is stated in more detail in the discussion, but it can be simply stated (one sentence) in the results. E. Figures and tables must be included that show the data generated by your experiment. Hand drawings and hand written calculations are acceptable, but they must be neat and labeled. Tables and figures must be numbered and titled. Table titles appear at the top, figure titles at the bottom. Tables: rows and columns are labeled. Figures: axes are labeled and contain units. VI. Discussion A. Restate in the first sentence or two the purpose and findings of the experiment. B. This section is the explanation of the results section. C. Include explanations of unpredicted or inconsistent results. D. Place the results into a setting. E. Compare and contrast results with existing knowledge. F. Explain why you think the results mean. G. Referencing other studies is appropriate.

8 H. Discussion should give the audience a general conclusion about the results and answers to the questions posed in the introduction. I. May refer to future experiments that can answer questions raised by this study. This is not always appropriate. What would you do next if you were to continue with this study? What would you change if you were to do the experiment again?

VII.

References

A. ALL thoughts, data or ideas that are not your own must be referenced. B. If something is a generally known fact, it does not need to be referenced. This includes, but is not limited to, Chemical molecular weights Species names Molecular composition of known compounds C. Be very careful when using websites. Information from the internet can be misleading or wrong, so you must be critical. Personal websites are not valid references. Any website used will be highly scrutinized, and if untrustworthy or even questionable, this will be detracted from your grade. D. The reference must be given in the text with the name of the author and the year of publication, and the full reference must be provided in the references section. The following selection taken from a recent article in EMBO reports (Richard and Pques, 2000) demonstrates acceptable methods of referencing others work: In human diseases, it is common to observe expansions of more than twice the original size. Such large expansions in yeast and humans could occur by successive rounds of unwinding/re-invasion of the donor sequence by the newly synthesized strand, allowing DNA synthesis to proceed more than once within the repeats (Pques et al., 1998; Figure 2c). An interesting case of large contraction of a CAG repeat was described during transmission of a myotonic dystrophy allele. O'Hoy et al. (1993) reported a large reduction of the number of CAG triplets associated with what they called a discontinuous gene conversion event. The resulting allele was a patchwork of both maternal and paternal alleles. Buard and Vergnaud (1994), Debrauwre et al. (1999) and Tamaki et al. (1999) also found complex recombination events in minisatellites. E. References must follow standard format. Examples given below.

9 Journal: Author, (year) title. Journal volume(issue), pages. ex.: Morehouse, S.I., Tung, R.S., Rodriguez, J.-C., Whiting, J.R. and Jones, V.R. (1993) Statistical evidence for early extinction of reptiles due to the K/T event. Journal of Paleontology 17(4), 198-209. Book: Author (year) title, number of pages. Edition number. Edition series, editor. Issue. Number of volumes. Publisher, city. ex.: Billoski, T.V. (1992) Introduction to Paleontology, 212 pp. 2nd ed. Trans. A. Translator. Series on Paleontology, edited by B.T. Jones, 6. 12 vols. Institutional Press, New York. Book with referred Chapter: Author (date) title. In: book editors (Eds), book title, edition pages. Volume. Number of volumes. Publisher, City. ex.: Grosjean, F.O. and Schneider, G.A. (1990) Greenhouse hypothesis: Effect on dinosaur extinction. Trans. M.A. Caterino. In: N.R. Smith and E.D. Perrault (Eds), Extinction, 3rd ed., pp. 175-189. Vol. 2. 5 vols. Barnes and Ellis, New York. Website: Author (date) Title of page or article (web site). ex.: Gutkind, J. S. (2000). Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors (http://www.stke.org).

VIII. Appendices A. Calculations B. Detailed information about equipment used if required. C. Answers to questions posed in the lab manual.

References Moriarty, M.F. 1997. Writing science through critical thinking. Jones and Bartlett Publishers, Inc., London. Richard, G.-F., and F. Pques. 2000. Mini- and microsatellite expansions: the recombination connection. EMBO Reports. 1:122-126.

10 EXPERIMENT NUMBER: 1

MICROSCOPIC MEASUREMENTS

1.1. INTRODUCTION The light microscope (Figure 1.1) employs visible light to detect small objects and it is probably the most well-known and well-used research tool in biology.

Figure 1.1. Diagram of a typical light microscope, showing the parts and the light path Types of Light Microscope The bright field microscope will be used in this course. Visible light is focused through a specimen by a condenser lens, and then is passed through two more lenses (objective and ocular lenses) placed at both ends of a light-tight tube (Figure 1.2). The latter two lenses each magnify the image. A third lens system located in the front part of the eye generates a real image on the retina. Limitations to what can be seen in bright field microscopy are related to resolution, illumination, and contrast. Resolution can be improved using oil immersion lenses, and lighting and contrast can be dramatically improved using modifications such as dark field, phase contrast, and differential interference contrast. Magnification, the degree of enlargement of the image of the object compared to its real size. It is provided by a two lens system; ocular lens (8 or 10X) and objective lens

11 (4, 10, 40 or 100X). Total magnification is the product of these two magnifying power values. Resolution, a measure of the clarity of an image. Resolving power is the ability of an optical instrument to show two objects as separate. For example, what looks to your unaided eye like a single star in the sky may be resolved as two stars with the help of a telescope. Any optical device is limited by its resolving power. Contrast, is the ability to determine same particular detail of specimen against its background. In a bright field light microscope, adjustable condenser with aperture diaphragm control or adding dyes may increase contrast of a transparent specimen. The maximum magnification obtained through a light microscope is 400X, in other words the closest two distinct points can be and still be resolved is 0.2 micrometer (m) about the size of the smallest bacterium (1 m = 10-6 m). This limitation is the result of light being diffracted by the object under observation and because diffracted light interferes with the image. Microscopic Measurements The linear size of a specimen observed in the microscope is best expressed in m and not as a total magnification. The actual size of an object viewed under the microscope can be estimated, based on the fact that the field of view for a given microscope and a given combination of lenses will have a constant diameter. A measuring device hemocytometer have been provided for you to measure the field of view.

0.05 mm

Figure 1.3. Hemocytometer under light microscope Focus on the SMALLEST SQUARES in the middle

0.05 mm

12 A device used for cell counting is called a counting chamber. The most widely used type of chamber is called a hemocytometer (Figure 1.3), since it was originally designed for performing blood cell counts. However, in this experiment hemocytometer is provided in order to measure the field of view. You can compute the size of the field for the higher magnification objectives as in the EXAMPLE below: Diameter of the field using X10 objective = 2 mm Diameter of the field using, X40 objective = 2 x 10/40 mm = 0.5 mm

1.2. MATERIALS light microscope hemocytometer microscope slides coverslips Pasteur pipettes and bulbs scalp hair elodea leaves safranin stain methylene blue ethanol isopropanol (to clean objective lenses)

1.3. PROCEDURE

1.3.1.Measurement of scalp hair diameter 1. Measure the visual field diameter with a hemocytometer. You should use both 10X and 40X objective lenses. Edge of the smallest square in the hemocytometer is 0.05 mm. Compare the two obtained values. Is there a meaningful proportion? 2. Clean a new glass microscope slide and coverslip with ethanol and tissue paper. 3. Pull out one hair from your scalp. Cut a piece shorter than the coverslip and place on the slide in the center. 4. Place a drop of water than the coverslip on the hair. 5. Observe your sample using 40X objective lens. To estimate the thickness of the sample, line up one edge of the hair against the edge of the visual field. Estimate the proportion occupied by the hair.

13 1.3.2. Observation of Elodea Leaf Cells Young leaves at the growing tip of elodea are particularly well suited for studying cell structure because these cells are only a few cell layers thick. 1. With a forceps, remove a single young leaf, mount it in a drop of water and cover with a coverslip. 2. Examine under 40X. 3. Add a drop of safranin stain to make the cell wall more obvious. Add the stain (1 drop) to one edge of the coverslip then draw the stain under the coverslip by touching a piece of tissue paper to the opposite site of the coverslip.

1.3.3. Observation of Epithelial Cells 1. Scrape the inside of your cheek with a flat toothpick. 2. Drop a methylene blue on a slide and stir the scraped epithelial cells in the stain. 3. Examine the preparation under 40X magnification. 4. Draw the two cell types you have examined and state the obvious differences between them. 1.4. QUESTIONS 1. What are the main differences between bright and dark field microscopy? Which type of samples better suit for bright and dark field microscopes, respectively? 2. Which part of the cell does safranin and methylene blue stain? 3. What are your estimates for the diameter of elodea and epithelial cells? Are these values consistent with the known ones?

1.5. REFERENCES 1. Figure 1.1: http://www.lmpc.edu.au/resources/Science/research_projects/light_microscope/light_micro scope.htm 2. Background information: http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html 3. Figure 1.3, modified from: http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html

14 EXPERIMENT NUMBER: 2

PLASMA MEMBRANE

2.1. INTRODUCTION The plasma membrane serves as the interface between the machinery in the interior of the cell and the extracellular fluid that surrounds all cells. It consists of a phospholipid bilayer together with proteins that can span the bilayer (integral/ transmembrane) or are peripherally attached to one face or the other (peripheral). The components of the bilayer frequently move laterally into other regions of the membrane, making it appear more fluid than static. This is called the fluid mosaic model of the cell membrane. In addition, proteins on the extracellular face can be heavily glycosylated, giving the membrane asymmetrical characteristics. The plasma membrane is selectively permeable; that is it will allow some substances to pass across it but not others. Molecules and ions move spontaneously down their concentration gradient (i.e., from a region of higher to a region of lower concentration) by diffusion. Molecules and ions can be moved against their concentration gradient, but this process, called active transport, requires the expenditure of energy (usually from ATP). Most membranes are permeable to water. Osmosis is a special term used for the diffusion of water through cell membranes. When the concentration of the solute is the same on the inside and outside of the cell, the water moves equally in both directions. The solution outside the cell is called isotonic. If the solution contains a higher solute concentration than the cell, it is hypertonic, but if it is lower, the solution is hypotonic.

2.2. MATERIALS light microscope cellophane tube tube clips starch solution iodine solution Elodea leaf NaCl soln.s lancet

15 2.3. PROCEDURE

2.3.1. Selective Permeability of an Artificial Membrane (CELLOPHANE) 1. Close one end of the cellophane tube with a clip. 2. Fill the tube about 3/4 full with starch solution. 3. Squeeze out the air and clamp the top with a second clip. 4. Wash the bag to remove any starch from the outside and blot dry with a tissue. 5. Submerge the bag in a beaker of iodine solution. Note the color of the bag and the color of the iodine solution. 6. After 30 minutes, remove the bag, rinse in water and blot dry. Note the color of the bag, and the iodine solution. 7. Report and explain your observations.

2.3.2. Osmosis in Elodea Leaf Cells & Human Blood Cells Slide 1:

Sample: elodea Solution added: none

Slide 2:

Sample: elodea Solution added: 0.1M NaCl

Sample: elodea Solution added: 0.3M NaCl

Slide 3:

Sample: elodea Solution added: 0.6M NaCl

Sample: blood Solution added: isotonic salt soln.

Slide 4:

Sample: blood Solution added: water

Sample: blood Solution added: 0.3M NaCl

Slide 5:

Sample: blood Solution added: 0.6M NaCl

Sample: elodea Solution added: unknown soln.

Sample: blood Solution added: unknown soln.

Figure 2.1. Split slides for Elodea and blood cells

16 1. Take 5 microscope slides, draw a perpendicular line dividing each slide into two parts and label the all 10 parts according to the sample and the solution to be added (Figure 2.1) with a permanent marker. 2. Observe them under 40X magnification 5 min after the solutions are added. 3. Note which solution induces shrinking or swelling. You should define the solutions in terms of hyper-, hypo- and isotonicity.

2.4. QUESTIONS 1. Can you estimate the concentration of XM NaCl? 2. Please define selective permeability of the plasma membrane. 3. What other functions can be attributed to the plasma membrane?

2.5. REFERENCES 1. Background information: http://darwin.nmsu.edu/~molbio/cell/PM.html

17 EXPERIMENT NUMBER: 3

CELLULAR FRACTIONATION

3.1. BACKGROUND INFORMATION Each organelle has characteristics (size, shape and density for example), which make it different from other organelles within the same cell. If the cell is broken open gently, each of its organelles can be subsequently isolated. The process of breaking open cells in an isotonic buffer is homogenization and the subsequent isolation of organelles is cellular fractionation. Isolating the organelles requires the use of physical chemistry techniques, and those techniques can range from the use of simple sieves, gravity sedimentation or differential precipitation, to ultracentrifugation of fluorescent labeled organelles in computer generated density gradients. In this experiment, cellular fractionation of a homogenized rat liver will be accomplished via a technique called differential centrifugation, which depends on the principle that as long as they are denser than the surrounding medium, particles of different size and shape travel toward the bottom of a centrifuge tube at different rates when placed in a centrifugal tube. Particles with higher density will sediment at a faster rate than the less dense ones. The first steps of differential centrifugation do not generally yield pure preparations of a particular organelle; so further steps are usually required. In many cases, further purification is accomplished by centrifugation of crude extract through sucrose density gradients. There are two types of centrifuges according to their rotors (centrifuge heads): Swinging-bucket model: rotors allow the tubes to swing out, causing the particles to move in a direction parallel to the walls of the tube. Fixed-angle model: the tubes are maintained at a particular angle, so that the particles sediment not to the bottom, but to the wall of the tube.

18 Relative centrifugal force (RCF) is defined as the ratio of the centrifugal force to the force of gravity, or RCF = Fc/Fg = 2r/980a

(expressed in radians/second) is converted to revolutions per minute (rpm) by substituting = (rpm) / 30, resulting in; RCF = 1.119 x 10-5 (rpm)2r where r is expressed in cm. RCF units are expressed as g.

3.2. MATERIALS Fresh rat liver 0.25 M sucrose 50 ml centrifuge tubes 3.3. PROCEDURE (Figure 3.1) Table-top and High-speed centrifuges Vortex Homogenizer Micropipettes and tips Ice bucket

1. Chop rat liver into approximate few mm3 pieces. 2. Add 0.25 M sucrose (10% w/v). 3. Homogenize with hand blender. 4. Centrifuge the homogenate to remove the cell debris at 800 g, 5 min. 5. Collect the supernatant: this is your whole homogenate. Save 5 ml for the next experiment
(Tube H) and record the volume of the rest.

6. Centrifuge the rest of the homogenate for 15 min at 5,000 g. 7. Resuspend the nuclear pellet in 0.25 M sucrose (save the suspension, Tube #2). 8. Centrifuge for 10 min at 24,000 g. 9. Resuspend the mitochondrial pellet in 0.25 M sucrose (save the suspension, Tube #3). 10. Rename Tube #2 as Tube N andTube #3 as Tube M.

19 3.4. QUESTIONS: 1. Why was liver chosen as the target organ for cellular fractionation? 2. Why was homogenization and subsequent suspension of organelles done in 0.25M sucrose solution? 3. Why were all steps carried out in relatively lower temperatures (centrifuges were fixed to 4C and organelles were stored at -70C)? 4. Please calculate the radius for centrifuge #1 using the given g value in the manual and the rpm value read from the centrifuge. 5. Please calculate the rpm values for centrifuge #2 using the given g and r values given in the manual. 6. Why do you think that the organelles obtained with this method are not absolutely pure? Can you suggest an additional or alternative method to better purify these organelles?

3.5. REFERENCES 1. Background information: http://homepages.gac.edu/~cellab/chpts/chpt3/intro3.html

20

Tube #1

homogenized rat liver in 0.25M sucrose 5 min

Centrifuge#1

800g

Tube #1
Cell Debris

Collect supernatant into a new tube

Tube #2

save 5 ml* (Tube H)

Whole Homogenate
5,000g 15 min
Centrifuge#2 r=9.4cm

Tube #2
Nuclear Pellet

(a) Collect supernatant in a new tube

Centrifuge#2 24,000g r=9.4cm

(b) Resuspend**

Tube #2

10 min

Nuclear Suspension*

Tube #3
Mitochondrial Pellet

(a) Resuspend**

Tube #3

Mitochondrial Suspension*

Figure 3.1. Schematic diagram of differential centrifugation

21 EXPERIMENT NUMBER: 4

ANALYSIS OF SUBCELLULAR FRACTIONS I

4.1. INTRODUCTION 4.1.1. Measurement of Succinate Dehydrogenase Activity Mitochondria enclose the biochemical machinery for cellular respiration; the aerobic processes by which sugars, fatty acids, and amino acids are broken down to carbon dioxide and water and their chemical energy captured as ATP. The Krebs cycle (also called as tricarboxylic acid or citric acid cycle) is a key series of reactions in this aerobic process (Figure 4.1). One of the best-studied enzymes in Krebs cycle is succinate dehydrogenase (SDH), which catalyzes the following redox reaction: Succinate + FAD Fumarate + FADH2

The objective of this lab is to measure the rate of the reaction in vitro using the mitochondrial fraction isolated in the previous lab session. The succinate fumarate reaction is measured by monitoring the reduction of an artificial electron acceptor, DCIP. DCIP is supplied as a dark blue solution and the color of this solution becomes lighter as DCIP is reduced. As the solution becomes lighter the absorbance at 600 nm decreases gradually which is observed via a spectrophotometer. To use an artificial electron acceptor, the normal path of electrons in the electron transport chain (ETC) must be blocked. This is accomplished by adding sodium azide to the reaction mixture. This poison inhibits the transfer of electrons from cytochrome a3 to the final acceptor, oxygen, so that electrons cannot be passed along: Succinate + FAD Fumarate + FADH2 eDCIP ETC Azide
SDH

FAD + 2H

Electrochemical gradient

blue

colorless

22

Figure 4.1. The Krebs cycle 4.1.2. Spectrophotometry Spectrophotometry is the measurement and analysis of electromagnetic radiation absorbed, scattered, or emitted by atoms, molecules, or other chemical species. The spectrophotometer operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The UV-Vis spectrum is from 200 nm to 800 nm. UV-Vis Spectrophotometry uses ultraviolet and visible electromagnetic radiation to energetically promote valence electrons in a molecule to an excited energy state. The UV-Vis spectrophotometer then measures the absorption of the energy to promote the electron by the molecule at a specific wavelength or over a range of wavelengths. The amount of electromagnetic radiation absorbed by a species

23 in a solution (A) depends on its concentration (c), the path length of the electromagnetic radiation (b), and the specific molar absorptivity (e) of the species: Beer-Lambert's Law A = ebc

The absorption of energy also depends upon the intensity of the incident light, Io, and the intensity of the exiting electromagnetic energy, I, where: A = log (I0 / I) There are five major components to a UV-Vis spectrophotometer; a radiation source, a wavelength selector or monochromator, a sample cell or cuvette, a detector, and a readout device.

Figure 4.2. The mechanism of spectrophotometer Transmittance (T) is another term that implies the amount of light energy that is transmitted.

%T = T x 100

A = - log10T

Cuvettes, or cells that contain the sample to be analyzed, should have parallel sides that are perfectly perpendicular to the radiation source. The cuvette used in UV-Vis spectrophotometry usually has a path length of 1 cm. The cuvette can be made up of several materials including fused silica, quartz, and plastic. Quartz cuvettes are transparent to UV and visible radiation and therefore are more commonly used for UV-Vis spectrophotometry. The cuvette must be wiped free of any marks or fingerprints before scanning. Any marks on the cuvette can scatter the UV-Vis radiation and cause error in the experiment. After each

24 scan the cuvette should be cleaned thoroughly. While scanning, the compartment holding the sample cell must be closed to all outside light sources. This prevents error in the experiment. 4.2. MATERIALS Mitochondrial fraction, Tube M from previous lab session Assay medium Sodium azide (0.04M) DCIP (5 x 10-4M) Malonate (0.2 M) Succinate (0.2 M) plastic cuvettes spectrophotometer micropipettes ice bucket

4.3. PROCEDURE 4.3.1. Measurement of Succinate Dehydrogenase Activity 1. Set the wavelength () of the spectrophotometer at 600 nm. 2. Label 10 tubes as shown in Table 5.1. Except for the ice-cold mitochondrial suspension (MS), all solutions should be at room temperature. 3. Prepare the MS for tube 7 by heating in microwave oven and then cooling on ice. 4. To all tubes, add the various solutions given in Table 4.1, except for MS in the stated order: Table 4.1. Contents of tubes to be prepared
Tube Order
Blank1 1 Blank2 2 Blank3b 3 4 5 6 7
a

Assay Medium (ml) 1st

3.7 3.2 3.1 2.6 3.4 2.9 2.7 3.4 3.4 2.9

Azide (ml) 2nd

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

DCIP (ml) Malonate (ml) 3rd

0.5 0.5 0.5 0.5 0.5 0.5 0.5

Succinate (ml) 5th

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

MSa (ml) 6th

0.3 0.3 0.9 0.9 0.6 0.6 0.6 0.6 0.6 0.6c

4th
0.2 -

Mitochondrial Suspension. bBlank3 serves as blank solution for both tube 3 and tubes 4,5,6 and 7. cThe MS

to be added to tube 7 will be heated

25 5. Add MS to Tube #1 and take the absorbance readings at t0. Repeat this step for each tube (Add MS and read the absorbance one by one for each tube, so that the absorbance reading can be obtained for t = 0 min).
Tube 1 Blank1 Tube 2 Blank2 Tubes 3-7 Blank3

6. At 5 min-intervals, measure the absorbance of the seven tubes for 35 min. Always remember to adjust for the 3 blanks as in step 6. Record all absorbance readings on DATA SHEET 1 (for t0, 5, 10, 15, 20, 25, 30 and 35). 4.4. QUESTIONS 1. On DATA SHEET 2, enter for each tube the total change in absorbance ( A) at each time interval. A(Tube n) = A(Tube n at ti) A(Tube n at t0) 2. Plot the reaction rates for tubes 1-6, in other words, A versus time (t0 graphs. 3. Plot the initial velocity (Vi) versus enzyme concentration ([E]) for tubes 1-3 (a single curve, which starts from the origin). Vi ( A/min) = A(Tube n at t35) A(Tube n at t0)/ t [E]for each tube = VolumeMS added 4. Comment on what happens to Vi when [E] has doubled and tripled. 5. What volume of mitochondrial suspension gives the highest initial velocity? Explain. 6. In tubes 4, 5, 6 and 7, four different factors that affect the rate of this reaction are used. State the nature of these factors and discuss their observed and expected effect on the reaction. 7. In the electromagnetic spectrum, where does the light with a of 600 correspond? Why do you think this was selected? Why was the absorbance reduced as the reaction preceded? 8. Malonate is a competitive inhibitor of SDH. What is the action mechanism of a competitive inhibitor in general? Do your results support this statement? 9. Krebs cycle contains a variety of enzymes. Why do we choose SDH, but not another enzyme for this experiment? NOTE: Include the data sheets and graphs in the results part.
References: 1. Background: http://www.easternct.edu/personal/faculty/adams/Resources/Lab3%20SDH.pdf
to 35).

Absolute values ! You may

either plot these 6 curves (not linear, draw as curves!) on a single graph or on 6 separate

2. Figure 4.1: http://www.personal.kent.edu/~cearley/PChem/pchem.htm

26 EXPERIMENT NUMBER: 5

ANALYSIS OF SUBCELLULAR FRACTIONS II

5.1. INTRODUCTION

5.1.1. Nucleic Acid To Protein Ratio Many molecules display maximum absorbance (Amax) at a particular wavelength (). This property may be used in identification of molecules. For nucleic acids; all four bases of DNA, accordingly the whole DNA molecule absorbs at = 260 nm. This Amax is used to determine the amount of both DNA and RNA. For proteins; Amax is reached at = 280 nm provided that the protein contains a normal percentage of tyrosine and tryptophan amino acids (aromatic rings). Proteins also show another Amax at a smaller in UV, due to their peptide bonds. In this experiment, the A260 values of the fractions collected by differential centrifugation will be measured. Assuming that An A260 of 1 is equivalent to 50 g/ml dsDNA, the DNA concentration in each fragment will be estimated.

5.1.2. Microscopic Examination Of Nuclear Fraction The nuclear fraction that has been isolated in the previous experiment (Tube N) will be examined microscopically to identify the nuclei, in addition approximate size of the nuclei will be measured (refer to Experiment #1). The nucleus is separated from the cytoplasm by an envelope consisting of two membranes. The entire chromosomal DNA is held in the nucleus; packaged into chromatin fibers by its association with an equal mass of histone proteins. The nuclear contents communicate with the cytosol by means of openings in the nuclear envelope called nuclear pores. Nucleoli are large, round or oval structures, in which ribosomal subunits are assembled, thus are rich in RNA and protein.

27

For the observations on nuclei, the stain to be used is aceto-orcein, which stains chromatin red. The nucleoli stand out since they do not stain with the orcein. Each nucleolus appears as a prominent, round, clear area.

5.2. MATERIALS Spectrophotometer Light microscope Micropipettes All fractions from Experiment #3 Aceto-orcein

5.3. PROCEDURE 5.3.1. Determination of DNA Concentration 1. Thaw all fractions and mix well. 2. Prepare 18 tubes and label as follows: H1, H2, H3, H4, H5, H6 N1, N2, N3, N4, N5, N6 M1, M2, M3, M4, M5, M6 for whole Homogenate for Nuclear pellet for Mitochondrial pellet

3. Prepare serial dilutions of each fraction by increasing the dilution ratio by 5.

Table 5.1. Serial Dilution steps for each fraction

Tube 1 2 3 4 5 6 1 1 1 1 1 0.25 0.25 0.25 0.25 0.25 0.25 M Sucrose( ml) Previous tube (ml) Volumefinal (ml) Final dilution ratio Volume left after dilution (ml) 1.00 1/5 1/25 1/125 1/625 1/3125 1.00 1.00 1.00 1.00 1.25

1.25 ml Stock Solution 1.25 1.25 1.25 1.25 1.25

28

4. Measure the absorbance values (A260) of all 24 tubes and determine the dilution factor for each fragment, which gives an absorbance between 0.2 and 1.5. 5. Try to calculate the DNA amount in each fraction by taking the dilution factors into account. DNA concentration (g/ml) = A260 x 50 g/ml x dilution factor

5.3.2. Microscopic Examination Of The Nuclear Fraction 1. Smear a tiny amount of the nuclear pellet on a clean slide with a spatula. 2. Immediately before the smear dries, add several drops of lacto-aceto-orcein. After 15 sec add a cover slip and press out the excess stain with a paper towel very gently. 3. Examine the preparation under the medium power objective. After locating a region with nuclei, switch to the high power objective. 4. Draw a typical nucleus, labeling nucleolus and estimate the nuclear diameter (refer to EXPERIMENT 1).

5.4. QUESTIONS 1. What does endosymbiotic theory suggest? 2. What was the reason that we have used plastic cuvettes in Experiment #4 and quartz cuvettes in this experiment? 3. The spectrophotometers cannot measure absorbance values higher than 2. Why?

29 EXPERIMENT NUMBER: 6 EXTRACTION OF DNA FROM BOVINE SPLEEN 6.1. INTRODUCTION 6.1.1. DNA Extraction Isolation of nucleic acids is the first step in most molecular biology studies. Extraction of nucleic acids from biological material requires cell lysis, inactivation of cellular nucleases and separation of the desired nucleic acid from cellular debris. Common cell lysis procedures include; Mechanical disruption (ex. grinding, hypotonic lysis), Chemical treatment (ex. detergent lysis) and Enzymatic digestion (ex. Proteinase K). The extraction medium usually contains an ionic detergent, which is required to lyse the nuclei and release the DNA. The detergent also inhibits any nuclease activity present in the preparation. Combination of phenol-chloroform and high concentrations of salt are often used to eliminate contaminants from nucleic acids. After cell lysis and nuclease inactivation, cellular debris may easily be removed by precipitation. Nucleic acids are usually precipitated with isopropanol or ethanol. 6.1.2. Gel Electrophoresis Nucleic acids may be separated electrophoretically on gel systems according to their size, shape and overall charge density (charge per unit of mass). This separation is commonly done on horizontal agarose gels. Due to their negatively charged phosphate backbone, nucleic acids move towards the anode in the electrical field. In the presence of ethidium bromide (EtBr), the separated nucleic acids are visualized under UV light. EtBr intercalates between the two strands of DNA. Electrophoresis is frequently used to determine size and purity of DNA. 6.1.3. Spectrophotometry The ratio of absorbance at = 260 to = 280 nm is one measure of the purity of a nucleic acid preparation. The 260/280 ratio of purified DNA is about 2. Higher ratio is often due to RNA contamination and lower values to protein contamination. 6.2. MATERIALS Bovine spleen Table-top centrifuge Spectophotometer quartz cuvettes Chilled blender Saline Citrate Buffer 2.6M NaCl Absolute ethanol Agarose 0.5X TEB 10X Loading buffer

30 6.3. PROCEDURE

6.3.1. DNA Extraction

homogenized bovine spleen (15g) in 150 ml cold SSC buffer dispersed in 4 centrifuge tubes

4,000 rpm
Materials soluble in physiological buffer (ex: RNA, many carbohydrates, some proteins)

15 min, 4C

DNP (DNA + Proteins), cell debris, unbroken cells

Add 40 ml of 2.6 M NaCl to each centrifuge tube. Vortex shortly to aid dissociation of the pellet. Shake vigorously till all pellet is dissolved. SALTING OUT 5,000 rpm
DNA dissolved in aqueous medium (salt solution)

20 min, RT

Proteins dissociated from DNA, cell debris, unbroken cells

Combine the supernatant of all 4 tubes in one beaker

Add 2X volume of absolute Ethanol

DNA: fish out with a sterile tip, air dry then incubate overnight at room temperature in Buffer TE (Tris-EDTA). Store at -20C.

31 Sections after 6.3.2. will be performed in the next lab!!!

6.3.2. DNA analyses with electrophoresis and spectrophotometry

Agarose Gel Electrophoresis 1. Prepare 1% agarose (w/v) in 35 ml of 0.5X TEB. Boil in microwave oven and let cool till about 55C. (Calculate: .......... g of agarose in 35 ml of 0.5X TEB) 2. Add 1.5 l Ethidium Bromide (EtBr) to agarose solution, mix and pour into the gel tray. (CAUTION!! EtBr is a potential carcinogen, do not touch or inhale!). 3. Let it polymerize for 10 min. 4. Prepare serial bovine DNA dilutions of 1:10, 1:100 and 1:1000. 5. Mix the samples (a control sample provided by the assistant and serial bovine DNA dilutions) with 6X loading buffer (glycerol + xylene cyanol + bromophenol blue) in 5:1 ratio on a piece of Parafilm and load the samples into sample wells. Run the gel for about 10 min. 6. Visualize the gel under UV light and record to the computer. Notice the differences between the mobility patterns of control and your samples. Try to explain this difference.

Spectrophotometry 1. Calculating the A260/A280 ratio will check the purity of the control and bovine DNA (Use 1:100 dilution of both control and bovine DNA). 2. Calculate the concentrations of both control and bovine DNA (refer to Experiment 5).

6.4. QUESTIONS: 1. Please explain the mechanism of salting out? 2. Can you suggest a method other than salting out to separate DNA from proteins?

32 EXPERIMENT NUMBER: 8 MITOSIS AND CYTOKINESIS 7.1. INTRODUCTION Recall that there are two basic cell types; prokaryotic and eukaryotic. Because of their simple genetic material, prokaryotes reproduce primarily as a result of fission; the splitting of a precasting cell into two, with each new cell receiving a full complement of the genetic material. In eukaryotes, the process of cell division is more complex, primarily because of the much more complex nature of the hereditary material, DNA (deoxyribonucleic acid) and the proteins complexed to it. In these cells, the genetic material is organized into chromosomes. Cell division usually involves 2 processes; mitosis (nuclear division) and cytokinesis (cytoplasmic division). Whereas mitosis results in the production of two nuclei, both containing identical chromosomes, cytokinesis ensures that each new cell contains all the metabolic machinery necessary for sustenance of life. Dividing cells pass through a regular sequence of events called the cell cycle. The majority of the time is spent in interphase and actual nuclear division-mitosis- is only a brief portion of the cycle. During interpase, the cell is scanning its environment for growth factors, producing new DNA, assembling proteins from amino acids, and synthesizing or breaking down carbohydrates. In short, interphase is a busy time in the life of a cell.

7.1.1. Mitosis in Onion Root Cells Cell divisions in plants localized in specialized regions called meristems. Meristems are regions of active growth. Plants have 2 types of meristems: apical and lateral. Apical meristems are found at the tips of plant organs (shoots and roots).

33

Figure 7.1. Stages of mitosis

7.2. MATERIALS light microscope previously prepared slide of onion root cells

7.3. PROCEDURE Obtain a prepared slide of a longitudinal section of an Allium (onion) root tip. This slide has been prepared from the terminal part of an actively growing root. It was "fixed" by chemicals to preserve the cellular structure and stained with dyes that have an affinity for the structures involved in nuclear division. Observe and draw cells undergoing different stages mitosis. Try to see the distinctive features of each stage.

34 Try to estimate the number of chromosomes of this organism. Find a cell undergoing cytokinesis. State how cytokinesis takes place in this tissue.

7.4. QUESTIONS Part I 1. Which part of the root was chosen for observing mitosis? Why? 2. Please estimate chromosome number for onion cells (2n=?). You should estimate this value through counting the number of chromatits observed in the metaphase plate or migrating in anaphase. 3. Which type of cyokinesis takes place in these cells? Part II 1. Intensity of DNA band is directly proportional to DNA concentration. Explain if your results are consistent with this fact. 2. Do you think that the bovine DNA is contaminated according to the A260/A280 ratio? If so, what may be the reason?

References: Figure 7.1 and background information: http://www.accessexcellence.org/RC/VL/GG/mitosis.html

35 EXPERIMENT NUMBER: 8 ANIMAL CELL CULTURE 8.1 INTRODUCTION 8.1.1 What is cell culture? The technique that is used to grow eukaryotic or prokaryotic cells in vitro (Latin: in glass) is called cell culture. Even though the term refers to a much larger scale of applications, in practice it almost exclusively refers to animal cell culture techniques. Since its establishment, animal cell culture techniques have allowed scientists to solve many puzzles on animal cell biology by allowing them to work under very strictly defined and controlled conditions. Moreover, its rapidity and cost efficiency together with complete control over the experiments makes this technique invaluable for scientists. Animal cell culture techniques are routinely applied in many laboratories today and allow the use of many applications formerly inapplicable or only possible by in vivo experiments. Such applications include:
Production of monoclonal antibodies Gene knock-out and over-expression studies Production of valuable proteins (i.e. enzymes and hormones)

Two basic findings lie in the core of cell culture technique are:
1. Cells can continue their growth under in vitro conditions as they can in their original tissues (as first shown by Ross Harrison in 1907[1]). 2. Cells can be frozen and stored for long periods of time and can still be viable after thawing.

Cell culture, very roughly speaking, consists of growing cells in vitro (plastic dishes), harvesting cells from plates, splitting and seeding cells on fresh plates and storing them by freezing when necessary (preferably in liquid nitrogen: -196o C). Animal cells are much more demanding than prokaryotic cells and thus their maintenance requires tedious and careful work, carefully chosen and prepared solutions and equipment, sterile and well conditioned environment as well as a carefully designed culture medium that mimics in vivo conditions. Selection of appropriate cell type among many is also crucial for the outcome of the experiment. 8.1.2 Source of Cells Primary cells: The cells are directly taken from the target tissues of organisms and added to the culture medium. Continuous cell lines: They are well established stable cell lines, mostly isolated from cancerous tissues. There are also some cell lines that are produced by manipulating their genome of a primary cell to overcome senescence; these cells are called immortalized. 8.1.3 Media In biology, the media used for various purposes can be classified into two categories: Defined media: The composition of the medium is known (i.e. which compounds are present and what are their concentrations) Undefined media: The composition of the medium is not known. Those media contain a component whose composition is not exactly known. For example the composition of yeast

36 extract, a component of Luria-Bertani (LB) broth, is unknown and thereby it renders LB broth undefined eventhough the exact composition of other ingredients are known. The most commonly used formula of an animal cell culture medium contains a basal medium and some other components that support the cell growth. There are defined and undefined media recipes for cell culture. Defined media for cell culture contains only basal media, whose composition is known; where as undefined media contains basal media and serum. Fetal bovine serum (FBS) is the most common type of serum used in cell culture and it is obtained from the fetuses of cows thus its composition is not exactly known. Basal media The selection of basal medium is crucial for the establishment of a healthy and well growing cell culture. Different cell types require different composition of substances and therefore different kinds of basal media for their optimal growth. There are many basal media types, such as Eagles medium and its derivatives (EMEM, AMEM, DMEM, GMEM, JMEM), Rosewell Park Memorial Institute medium derivates (RPMI 1629, RPMI 1630, RPMI 1640), Fischerss, Liebovitz, Trowell, and Williams media, CMRL 1060, Hams F10 and derivatives, TC199 and derivatives, MCDB and derivatives, NCTC and Waymouth [2]. Components of basal media [2]
1. Balanced salt solution: They contain inorganic salts to maintain physiological pH and osmotic pressure, and to provide necessary ions that are used for key metabolic activities (membrane potential, cofactors) (Na+, K+, Mg2+, Ca2+, Cl-, SO44+, PO43+, and HCO3-). 2. Buffering system: Mostly used media are buffered with bicarbonate ions. HCO3- ions react with CO2 formed by the cells through oxidative respiration and also with CO2 supplied in the atmosphere. There are different buffering systems which use high concentrations of PO43+ or beta-glycerophosphate and low bicarbonate. It is also possible to use organic buffers such as HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid). 3. Energy source: Glucose. Other sugars can be used (i.e. maltose, sucrose, fructose, galactose and manose). 4. Amino acids 5. Vitamins 6. Hormones and growth factors (they are normally present in serum but in defined media they are added to the basal medium) 7. Proteins and peptides 8. Fatty acids and lipids 9. Accessory factors (zinc, iron, copper, selenium) 10. Antibiotics

Serum [2]
1. 2. 3. 4. 5. Growth factors Albumin Transferrin Anti-proteases Attachment factors

8.1.4 HEK 293T cell line HEK 293T or simply 293T cell line is a highly transfectable derivative of human embryonic kidney 293 cells. 293 cells were obtained from and aborted fetus kidney cells by

37 Frank Graham in late 70s [3,4]. Those cells were transfected with sheared adenovirus 5 DNA. At first the effect of this transfection at the genomic level was unknown, but it was observed that these cells overcame senescence, in other words immortalized. In 1997, Nathalie Louis and her colleagues found out that approximately 4.5 kilobases of DNA from the left arm of the viral genome was inserted into chromosome 19 [5]. In addition to adenovirus 5 DNA, 293T cells are also transfected with simian virus 40 (SV40) large T antigen is incorporated into the genome [6]. 8.1.5 Reporter Gene Reporter gene is basically a gene that produces a detectable product or observable effect when introduced into the cell. Reporter genes are generally attached to other genes of interest and used to see if the transfection procedure has been successful or to measure the activity of a specific gene. Green fluorescent protein (GFP), luciferase and -galactosidase are common examples. 8.1.6 Vector Vectors are molecular carriers that are used to introduce foreign DNA into the prokaryotic or eukaryotic cells. Plasmids and viruses are two types of vectors used in molecular biology. Plasmid vectors contain four important sites:
An origin of replication A genetic marker A multiple cloning site Promoter site(s)

Every plasmid has to have an origin of replication since it is required for plasmid, and thereby the DNA of interest that is inserted into the plasmid, to replicate. Genetic markers allow us to select the successfully transfected cells. Antibiotic resistance genes are commonly used in cell culture as genetic markers. The DNA fragment of interest is inserted into the multiple cloning site. This site contains many recognition sites for different restriction enzymes thereby allowing us to use a suitable enzyme for our experiment. In eukaryotes, the promoter sequence is a part of gene regulation. For a gene to be transcribed, the appropriate transcription factor must bind to the promoter region and turn the gene on. Different promoters differ in activity; some promoters are highly active (i.e. CMV) where as some are not. It is possible to construct plasmids with different promoter regions and thereby altering its activity.

38

Figure 8.1. Map of pcDNA3 from Invitrogen Life Technologies.

8.1.7 Green Fluorescent Protein Green fluorescent protein is isolated from the jellyfish Aequorea Victoria. Thanks to its unique shape, this protein fluoresces green when excited by blue light. GFP is 238 amino acids length and has a unique structure comprised of 11 -barrel and a single alpha helix which contains the chromophore [7, 8]. To visualize GFP a special type of microscope, namely inverted fluorescent microscope is used. This microscope is capable of exciting the cells with light at specific wavelengths by using special filters and visualizing fluorescence emitted by the cells. Its light source and lenses are inverted for better focusing.

Figure 8.2. An inverted light microscope.

39 Introducing foreign DNA into cells There are various techniques to introduce foreign DNA into cell. These techniques include calcium phosphate, electrophoration, heat shock, transduction using viruses that are incapable of replicating, via commercial transfection reagents etc. For the introduced DNA to be translated, it has to enter the nucleus. The majority of the foreign DNA is degraded and only a small proportion reaches the nucleus. In the nucleus, it may stay free in the plasmid form or it may be inserted into the hosts genome. If it remains free as a plasmid it is called a transient transfection because the transfected DNA can be lost as the cells proliferate. If the transfected DNA is inserted into the hosts genome it is called a stable transfection and it will remain in the hosts genome regardless of proliferation. 8.2. MATERIALS 6 well culture plates (Figure 8.3), Inverted Microscope (Figure 8.2) Plasmid DNA pEGFP-C2 Micropipets, Sterile tips and tubes (Autoclaved) DMEM (Dulbecco/Vogt Modified Eagle's Minimal Essential Medium) 2M CaCl2, 2X HEPES Buffer 8.3. METHODS 1. Start with 293T cells cultured on 6 well plates (0.8x106 cells/well) with 1 ml of DMEM added. 2. Prepare the transfection solutions in 6 tubes (1.5ml centrifuge tubes) as indicated on Table 8.1 and in Figure 8.3. Wells 1 and 2 will be negative controls; add H2O instead of plasmid DNA to well 1 and add empty plasmid (i.e., plasmid with no reporter gene) to well 2. You will add 0.1 g plasmid to wells 3 & 4 and 1 g to wells 5 & 6, respectively. 3. Mix the tubes by tapping after addition of both CaCl2 and HEPES. 4. Wait growth of DNA/Ca-P particles for 5 min after all contents of the transfection solution is mixed. 5. After 5 min, spread the DNA/Ca-P onto the wells by pipeting all over each well. 6. Incubate cells with transfection solution at 37C overnight. 7. Next Day Evaluation of transfection with inverted microscope Inverted Microscope at AKIL Lab, BE ON TIME ! Check and take picture of your cells both with and without florescence filter. - Without Filter all cells are visible - With Filter only cells transfected with pEGFP-C2 is visible. Table 8.1. Components of transfection solutions H2 O H2O or Plasmid DNA 2M CaCl2 2X HEPES Total Volume Spread on each well for 6 well plates (solutions in l) for 1 well 209.5 10.0 30.5 250.0 500.0 500

40

Figure 8.3. Schematic representation of a 6-well culture plate. Wells are numbered and labeled according to the kind of plasmid DNA added. Questions
1. What is the advantage of using in vitro techniques than in vivo techniques? 2. Why does the transfected DNA have to enter the nucleus to be translated? 3. Why the selection of the appropriate cell type (e.g. kidney cell, fibroblasts, neuronal cells etc) is crucial for the experimental design and results 4. Please shortly explain two of the transfection methods (you can use the ones mentioned in the lab manual or find different ones in the books/ on the internet). 5. What is a defined medium? Undefined medium? Why it is preferred to use defined media instead of undefined media? Bonus 1. Eukaryotic cells, like prokaryotic cells, have a defense mechanism against the foreign DNA. They recognize and destroy any piece of DNA if it is detected as not of the cells own. Even though in the cell lines used for transfection this defense mechanism is intact, it is still possible to successfully introduce foreign DNA into the cell. Propose a rational explanation for this phenomenon.

References
[1] Harrison, R. G. (1907). Proc. Soc. Exp. Biol. Med., 4, 140 [2] Basic Cell Culture: A Practical Approach. J. M. Davis. IRL Press at Oxford University Press, Oxford, 1994 [3] HEK Cell, 24.09.2007 - http://en.wikipedia.org/wiki/HEK_cell [4] Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Graham FL, Smiley J, Russell WC, Nairn R. J Gen Virol. 1977 Jul;36(1):59-74. [5] Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. Louis N, Evelegh C, Graham FL. Virology. 1997 Jul 7;233(2):423-9. [6] LGC Promochem: Cell Biology Collection, 24.09.2007 - http://www.lgcpromochematcc.com/common/catalog/numSearch/numResults.cfm?atccNum=CRL-11268 [7] Tsien R (1998). "The green fluorescent protein". Annu Rev Biochem 67: 509-44. [8] Green Fluorescent Protein, 24.09.2007 - http://en.wikipedia.org/wiki/Green_fluorescent_protein

41 EXPERIMENT NUMBER: 9 ANALYSIS OF POLYSACCHARIDES

9.1. INTRODUCTION Monosaccharides A molecule consisting of C, H, and O in a 1:2:1 ratio [(CH2O)n]. Most monosaccharides in protoplasm are either 3-carbon sugars (trioses), 5-carbon sugars (pentoses) or 6-carbon sugars (hexoses). The sugars are also characterized by whether they contain an aldehyde (aldoses) or ketone group (ketoses). Polysaccharides Many different kinds of polysaccharides are known, but starch, glycogen and cellulose are especially important in living systems. All three are made up entirely of glucose subunits joined by the removal of water (condensation) to form glycosidic bonds. They differ strikingly in their structural and chemical properties. Table 9.1. Properties of three important polysaccharides Type Produced by Function Glycogen Animal cells Starch Plant cells Structural polysaccharide that comprises the bulk of plant cell walls -glycosidic bonds (as are not easily broken in nature, cellulose is a very tough and durable substance) Cellulose

Storage polysaccharides that may be deposited in large granules in cells

Type of glycosidic bond

-glycosidic bonds (glycogen is more highly branched than starch)

Hydrolysis Breaking of the glycosidic bonds involves the adding back of water across the bond and thus termed hydrolysis. This can be achieved by heating the polysaccharide in the

42 presence of water and/or strong acids. Enzymes catalyze the same hydrolytic reaction in normal aqueous solutions without the extreme conditions.

Controls in an Experiment A negative or a positive control is in a protocol in order to make sure that the result obtained is not due to; A protocol that is not capable of producing a positive result (systematic error), An experimental error in the course of performing the protocol. In the positive control, a positive effector instead of the tested variant is included, whereas in a negative control the tested variants are not included and a negative result is expected.

9.2. MATERIALS 1M glucose 0.5% starch Benedict's solution Amylase toothpicks (a pack) test tubes Pasteur pipettes 6M HCl 5M NaOH Na2CO3

9.3. PROCEDURE

9.3.1. Part I 1. Collect as much saliva as possible in the beaker provided. 2. Label 3 tubes as A, B, C, D. Proceed with the experiment as stated in Table 9.2. Table 9.2
Tube A 15 ml H2O + 30 drops saliva Tube B Tube C 30 drops saliva + 15 ml 15 ml H2O + 30 drops starch starch Incubate at 37 for 1 hr Add 20 drops Benedict's solution (after 1 hr) Heat for 5 min in a boiling water bath Observe and note the color changes Tube D

15 ml starch + amylase

9.3.2. Part II 1. Label 4 tubes as E, F, G, H. 2. Proceed with the experiment as stated in Table 8.3.

43 Table 9.3 Tube E

15 ml H2O

Tube F
15 ml starch soln.

Tube G
One toothpick broken into tiny pieces + 15 ml H2O

Tube H
15 ml glucose soln.

Heat in a boiling water bath for 15 min and let cool Add 20 drops of Benedict's solution to all the tubes Note the colors Heat in a boiling water bath for 5 min Note the colors

9.3.3. Part III 1. 2. Label 3 tubes as I, J, K, L. Proceed with the experiment as stated in Table 8.4.

Table 9.4 Tube I

15 ml H2O + 10 ml 5N HCl

Tube J

Tube K

Tube L
15 ml glucose soln. + 10 ml 5N HCl

One toothpick broken 15 ml starch solution + into tiny pieces + 10 ml 10 ml 5N HCl* 5N HCl Heat in a boiling water bath for 15 min and let cool

Add Na2CO3 gradually, untill bubble formation ceases Add 20 drops of Benedict's solution Heat in a boiling water bath for 5 min Note the colors * CAUTION! You should always add acid to water, NOT WATER TO ACID! ** CAUTION! Point the mouth of the tube away from you. *** CAUTION! Do not add much NaOH, as the reaction proceeds very rapidly.

9.4. QUESTIONS 1. For EACH part of the experiment state; Negative control tubes Positive control tubes Test tubes 2. What does Benedicts solution contain? Which reaction does it undergo?

44 3. Why did we heat the tubes after adding Benedicts solution? 4. Why is glucose called a reducing sugar? 5. For Part I, what can you conclude about your saliva? What would you expect the result to be if you had used the toothpick instead of starch solution in the experiment? 6. Please write the reaction that results in bubble formation when Na2CO3 is added in part. Why do we need this reaction? 7. Draw the structures of, glucose, glycosidic bonds in starch and cellulose the hydrolysis reactions

45 EXPERIMENT NUMBER: 10

CELLULAR CARBOHYDRATES

10.1. CELLULAR CARBOHYDRATES Carbohydrates are a group of macromolecules, which includes simple sugars, and all larger molecules constructed of sugar subunits. They mainly function as energy store-houses and as durable building materials. The glycogen in animals and the starch in plants serve as energy stores. Other carbohydrates, such as chitin and cellulose, have structural roles in animals and plants, respectively. Oligosaccharides are components of the glycoproteins and glycolipids found in the plasma membrane. These sugar chains, which always face the cell's exterior, stabilize the position of glycoproteins and glycolipids within the membrane, function in cell adhesion, and confer immunological specificity to the cell surface. Cytology is the study of cells. Every class of macromolecules can be localized in cells with specific cytochemical reactions. The term cytochemistry can refer to any methodology that probes the chemical nature of the cell but usually is reserved for specific staining, reactions and subsequent microscopic analysis. An important cytochemical method used to identify cellular carbohydrates is the Periodic Acid-Schiff (PAS) Reaction. The reaction stains insoluble polysaccharides (glycogen, starch, cellulose), and the objects of this experiment are: to carry out the PAS reaction on fixed blood smears and to localize PAS-positive material in leukocytes

10.2. HUMAN BLOOD Human blood is composed of blood cells, cell-like components and macromlecules suspended in a clear, staw-colored liquid called plasma. Figure 7.1 shows the components of blood when it is treated with an anti-coagulant and centrifuged.
Figure 10.1. Components of human blood

46 10.2.1. Blood Cells Blood cells are produced in the bone marrow and arise from a single type of cell called a pluripotent stem cell.

Figure 10.2. Types of human blood cells 10.2.2. Blood Cell Types Histologists frequently use Wright-Giemsa stain for the identification and study of human blood cells. Wright-Giemsa stain is a mixture of methylene blue, methylene azure, and the eosinates of blood. The azures act as bases and stain the basophilic (base loving) elements of the cells blue, while the eosins behave as acids and stain the acidophilic (acid loving) structures red. Since there is a combination of dyes and a varying affinity for each, the various parts of the cell are stained in hues of pink, purple, blue, and red. Figure 10.2 is a diagram of the various human blood cell types as they appear in a fixed smear. There are two classes of cells, red blood cells/erythrocytes and white blood cells/leukocytes. The leukocytes are placed into two groups, the granulocytes, which have conspicuous cytoplasmic granules, and the agranulocytes, which lack them. The granulocytes include the neutrophils, eosinophils, and basophils. The agranulocytes include the

47 lymphocytes and monocytes. In addition to these cell types there are the platelets, which are small cell fragments. Erythrocytes: The erythrocyte is the most abundant (about 5 x 10 per l) and the smallest blood cell type. Human erythrocytes lack a nucleus and are biconcave. In WrightGiemsa-stain preparations, they appear pink with a central region staining lighter because of the concavity. They lack any internal organelles but are filled with hemoglobin for the function of oxygen transport. Leukocytes: Table 10.1 Table 10.1. Types and properties of human leukocytes Leukocytes
about 7 x 103/l leukocytes in human blood GRANULOCYTES Neutrophils % among leukocyte population Cell morphology 55-75% Eosinophils 1-5% Basophils 0.5% AGRANULOCYTES Lymphocytes 20-40% Monocytes 5-7% Large, round or oval, some have blunt pseudopods Round or kidneyshaped, stain lightly and do not contain small, lilacstained granules

Round

Round

Round

Round Round and large, often with indentation. They stain dark purple, and there are usually clumps of chromatin present

Morphology of the nucleus when stained

Dark purple, multilobed nucleus

Dark purple nucleus with two large lobes connected by a thin strand of chromatin

Stain purple, may be round, indented, banded, or lobed Contain numerous large, dark purple basophil granules

Morphology of the cytoplasm when stained

Pale pink with lightly stained granules

Filled with many large, reddish orange eosinophil granules

Stains blue

Function

Body's first defense against invading microorganisms

Selectively phagocytize foreign proteins that are complexed with antibodies

Involved in allergic reactions

Production of antibodies in the immune response

Act as macrophages in tissues where they phagocytize a variety of foreign substances

48 Platelets: Platelets are small, irregularly shaped cell fragments that have broken away from megakaryocytes in the bone marrow. Platelets are cell fragments produced from megakaryocytes. Blood normally contains 15-45 x 104 platelets per l. They range 1-4 m in size and stain blue or purple. These numerous cell fragments play a major role in the blood clotting process.

10.3. PERIODIC ACID-SCHIFF REACTION (PAS) The periodic acid-Schiff Reaction was first introduced for histological preparations by J.F.A. McManus in 1946. It is used to stain glycoproteins, polysaccarides, certain mucopolysaccarides, glycolipids and certain fatty acids in tissue sections. 1. The first step in the PAS reaction is treatment of the fixed cells with periodic acid (HIO4). HIO4 oxidizes the 2,3-glycol grouping of sugars to a dialdehyde (Figure 10.3). The reaction is very specific, as it forms aldehydes within the polysacchride molecule but it does not continue the oxidation of the polymers to low molecular weight water soluble forms. 2. The preparation is then stained with Schiff's reagent, a colorless liquid. Schiff's reagent reacts with aldehydes to form colored compounds (purplish red). Any cell structures that stain purplish red with PAS are said to be PAS-positive. 3. It is important to note that PAS reaction stains insoluble sugars, which contain 2,3glycol grouping.

10.3.2. Slide Preparation You will be provided with two blood smears that have previously been fixed in a 9:1 mixture of ethanol: formalin. In cytological preparations, cells must be fixed so that their general morphology, and internal structures will be preserved. If the cells were not treated with a fixative, the hydrolytic enzymes of the lysosomes would be released and, eventually, much of the cell would be digested. An effective fixative must render cell components insoluble, last they be washed out during subsequent treatment. It should also prevent subsequent swelling or shrinkage of the cell contents. Often, the fixative improves staining by enhancing the affinity of the cell components for dye molecules. Many fixatives contain an alcohol plus one or more of the following: acetic acid. formalin, chloroform.

49 For the various steps in the PAS reaction, the fixed blood smears will be transferred to or stored in Coplin Jars. As the slides are being processed, the slide surface with the cell preparation must never be allowed to rest against another slide. You will work with two fixed blood smears, one for the PAS reaction and one for the diastase control. After the control slide is treated with diastase, both slides are treated with HIO4 followed by Schiff's reagent. The staining Jar should be kept in the dark since Schiff's reagent deteriorates in the light. After treatment with Schiffs reagent, the slides are rinsed with a bisulfite bleaching solution. This rinse assures that the red coloration is due to the cytochemical reaction and not due to the presence of any basic fuchsin that may be formed during the staining procedure. Subsequently, the slides are counterstained. Only half of each blood smear will be counterstained, so that there will be a portion of each smear stained only by the PAS reaction. The final step in the procedure is the mounting of a coverslip. The coverslip protects the cell preparation and allows immersion oil to be removed easily.

50

Figure 10.3. The steps in the PAS reaction, shown here a staining portion of a glycogen molecule

(a)

(b)

(c)

Figure 10.4. The steps for preparation of a thin blood smear

51 10.4. MATERIALS Coplin jars 200 ml 9:1 ethanol:formalin fixative dispensed in five Coplin Jars (prepare shortly before use): 180 ml absolute ethanol + 20 ml formalin (i.e., 37% w/w formaldehyde) 300 ml 1% periodic acid solution (good for several weeks if stored in a dark bottle) dispensed in eight Coplin Jars: dissolve 3.0g H5IO6 in 300 ml dH2O 100 ml 1N HCl add 8.1 ml concentrated HCl into 91.9 ml dH2O (in a fume hood) 220 ml Schiff's reagent dispensed in Coplin jars 200 ml Bleaching solution: 180 ml distilled water, 10 ml 10% Na or K metabisulfate solution, 10 ml 1N HCl 100 ml of 10% metabisulfite solution: dissolve 10g Na or K metabisulfite in dH2O 10.5. PROCEDURE NOTE: All staining procedures are done in Coplin jars; use a zigzag arrangement of up to nine slides per jar. Always use forceps to place slides in or to remove slides from Coplin Jars. 1. Label the slides with your initials.!!! The markings will also identify the slide surface with the cell preparation. 2. Smear a tiny drop of blood on the slides. Prepare the blood smear as shown in Figure 10.4. The smear must be very thin and the boundaries should not exceed the cover slip length. Let the blood on the slides to dry for 8-10 min. 3. Incubate slides in ethanol:formalin fixative for 10 min. 4. Place slides in periodic acid for 10 min. 5. Rinse the slides under running tap water for 1 min (in a Coplin Jar) and then rinse with distilled water, as in step 3. 6. Place the slides in Schiff's reagent (in a fumed hood) for 10 min. The staining jar should be kept in the dark. 7. Remove the slides from the staining jar with forceps and transfer them to a clean Coplin jar. Rinse the slides under running distilled water for several seconds.

52 8. In a fume hood, replace the distilled water with freshly prepared bisulfate bleaching solution. Decant after 2 min and repeat this bleaching procedure 2 more times. 9. Rinse the slides under running tap water for 5 min and then in distilled water. Wipe the back of each slide with a tissue paper and allow to air-dry in a vertical position. 10. When the slides are completely dry, place a cover slip and observe using 40X and the 100X objective lens.

10.6. QUESTIONS 1. Why do you think the PAS reaction does not stain nucleic acids? 2. Why do we need to fix cells before further cytochemical techniques? References: 1. PAS reaction: http://stainsfile.info/StainsFile/stain/schiff/reaction-pas.htm 2. Blood cells background information and Figure 10.2: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html