TECHNICAL MANUAL

VERSION 1.1.0

In Vitro Proliferation and Differentiation

of Rat Neural Stem And Progenitor Cells (Neurospheres)
Using NeuroCult

Table of Contents
1.0 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Storage of Medium and Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 Additional Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
1
1

2.0 Preparation of Complete NeuroCult NS-A Proliferation Medium (Rat) . . . . . . . . . . . . . . 2

2.1 Thawing of Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Preparation of Medium, Supplements and Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1 Preparation Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2
2
2

3.0 Preparation of E18 Rat CNS Cells for In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1 Preparation of Primary Rat CNS Tissue and Initial Culture of Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . .

3.2 Preparation of Rat Cells from Cryopreserved Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2.1 Thawing Cryopreserved Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2 First Passage of Cryopreserved Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4
4
4

4.0 Subculture of Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5.0 Differentiation of Rat Neurospheres into Neurons, Astrocytes and Oligodendrocytes . . . . 6
5.1 Preparation of NeuroCult NS-A Differentiation Medium (Rat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Differentiation of EGF- and/or FGF-b-Responsive Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1 Preparation of Poly-L-Ornithine Coated Glass Coverslips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3 Differentiation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6
6
6
7

6.0 Immunolabeling Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

6.1
6.2
6.3
6.4
6.5

Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Blocking and Labeling with Primary Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Secondary Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7.0 Representative Photographs of Cultured Rat Neurospheres . . . . . . . . . . . . . . . . . . . . . . . . . 11

8.0 Representative Photographs of Immunolabeled Differentiated Rat Neurospheres . . . . . . . 13

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ii

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1.0 Materials
1.1 Storage of Medium and Supplements
Very important! Upon arrival, cryopreserved neurospheres* must be stored IMMEDIATELY at -135C or colder, or in liquid nitrogen.
NeuroCult NS-A Basal Medium (Rat)* should be stored at 2 - 8C. NeuroCult NS-A Proliferation Supplements (Rat)* and NeuroCult
NS-A Differentiation Supplements (Rat)* should be stored at -20C.
After NeuroCult NS-A Proliferation Supplements (Rat) have been added to the NeuroCult NS-A Basal Medium (Rat), storage at
2 - 8C is recommended for no more than 1 month. Long-term storage at 2 - 8C is not recommended. Refer to Section 2.0 for
preparation of Complete NeuroCult NS-A Proliferation Medium (Rat).
After NeuroCult NS-A Differentiation Supplements (Rat) have been added to the NeuroCult NS-A Basal Medium (Rat) to make "Complete"
NeuroCult NS-A Differentiation Medium (Rat), storage at 2 - 8C is recommended for no more than 1 month. Long-term storage of the
"Complete NeuroCult NS-A Differentiation Medium (Rat) at 2 - 8C is not recommended.

1.2 Equipment Required

Vertical laminar flow hood (e.g. Canadian Cabinets) certified for Level II handling of biological materials
Low speed centrifuge (e.g. Beckman TJ-6)
37C incubator with humidity and gas control to maintain >95% humidity and an atmosphere of 5% CO2 in air
(e.g. Forma 3326)
Vortex (e.g. Vortex Genie)
Pipette-aid (e.g. Drummond Scientific)
Hemacytometer (e.g. Brightline)
Forceps
Routine light microscope for hemacytometer cell counts
Inverted microscope with flatfield objectives and eye pieces to give object magnification of approximately 20 - 30X, 80X,
and 125X (e.g. Nikon Diaphot TMD)
T-25 cm2 tissue culture flasks (Nunc Catalog #156367 or VWR Catalog #15708-130)
8-well culture slide (BD BioCoat 8-well culture slide) pre-coated with Poly-D-Lysine/Laminin (BD Catalog #354688) or
Poly-D-Lysine (BD Catalog #354632) OR coverslips (Fisher Scientific Catalog #12-545-82)
24-well culture dish (Corning Catalog #3526 or equivalent)
Conical tubes, 14 mL (Falcon Catalog #352001)

1.3 Additional Reagents Required

Recombinant Human Epidermal Growth Factor (rh EGF; Catalog #02633)
Recombinant Human Fibroblast Growth Factor, basic (rh FGF-b; Catalog #02634)
Heparin 0.2% in PBS (Catalog #07980)
Dulbecco's PBS without Ca++ or Mg++ (Catalog #37350)
Trypan Blue (Catalog #07050)
10 mM Acetic Acid (required for dissolving rh EGF powder)
Bovine Serum Albumin (required for dissolving rh EGF and rh FGF-b powder)
2% Glucose in PBS
Triton X-100 (Sigma Catalog #T9284)
Poly-L-Ornithine (Sigma Catalog #P3655)
70% Ethanol
4% Paraformaldehyde
* Sold under license from StemCells California, Inc. US Patent Nos. 5,750,376; 5,851,832; 5,980,885; 5,968,829; 5,981,165; 6,071,889; 6,093,531; 6,103,530; 6,165,783; 6,238,922
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2.0 Preparation of Complete NeuroCult NS-A Proliferation Medium (Rat)

2.1 Thawing of Supplements
Bottles of NeuroCult NS-A Proliferation Supplements (Rat) should be thawed overnight at 2 - 8C (in the refrigerator) or for 1 - 2
hours at 37C before addition to the NeuroCult NS-A Basal Medium (Rat). The NeuroCult NS-A Proliferation Supplements (Rat) can
be aliquoted into 10 mL volumes and stored at -20C until required for use. However repeated thawing and freezing is not
recommended.
Thaw one bottle (50 mL) of NeuroCult NS-A Proliferation Supplements (Rat) as described. Add the entire volume of NeuroCult NS-A
Proliferation Supplements (Rat) to one bottle (450 mL) of NeuroCult NS-A Basal Medium (Rat) or add 1 mL of NeuroCult NS-A
Proliferation Supplements (Rat) to every 9 mL of NeuroCult NS-A Basal Medium (Rat) (1/10 dilution).
OR
Add 1 mL of NeuroCult NS-A Proliferation Supplements (Rat) to every 9 mL of NeuroCult NS-A Basal Medium (Rat) (1/10 dilution).

2.2 Preparation of Medium, Supplements, and Growth Factors

A stock solution of 10 g/mL of rh EGF (Catalog #02633; powder at 200 g/vial) is made up by adding 0.1 mL of sterile 10 mM acetic
acid containing at least 0.1% bovine serum albumin (BSA) to initially dissolve the rh EGF powder and then adding 19.9 mL of
NeuroCult NS-A Basal Medium (Rat) containing NeuroCult NS-A Proliferation Supplements (Rat). The stock solution of 10 g/mL of rh
EGF should be stored as 0.5 mL aliquots at -20C until required for use. Add 2 L of 10 g/mL rh EGF to every 1 mL of NeuroCult
NS-A Basal Medium (Rat) containing NeuroCult NS-A Proliferation Supplements (Rat), to give a final concentration of 20 ng/mL of rh
EGF. Mix well.
A stock solution of 10 g/mL of rh FGF-b (Catalog #02634) is made up in PBS and 0.1% BSA and stored as 0.5 mL aliquots at -20C.
Add 1 L of rh FGF-b to every 1 mL of NeuroCult NS-A Basal Medium (Rat) containing NeuroCult NS-A Proliferation Supplements
(Rat) and rh EGF to give a final concentration of 10 ng/mL of rh FGF-b. Mix well.
Store the stock solution of Heparin (Catalog #07980; 0.2% in PBS) at 2 - 8C in small aliquots (0.5 mL). Add 1 L of 0.2% Heparin
solution to every 1 mL of NeuroCult NS-A Basal Medium (Rat) containing NeuroCult NS-A Proliferation Supplements (Rat), rh EGF
and rh FGF-b to achieve a final concentration of 0.0002% (2 g/mL).
"Complete" NeuroCult NS-A Proliferation Medium (Rat), containing NeuroCult NS-A Basal Medium (Rat), NeuroCult NS-A
Proliferation Supplements (Rat), 20 ng/mL rh EGF, 10 ng/mL rh FGF-b and 2 g/mL heparin is now ready to use.

2.2.1 Preparation Summary

For every 1 mL of NeuroCult NS-A Basal Medium (Rat) containing NeuroCult NS-A Proliferation Supplements (Rat) add:

Component

Volume added per mL

Final Concentration

10 g/mL rh EGF

2 L

20 ng/mL

10 g/mL rh FGF-b

1 L

10 ng/mL

0.2% Heparin

1 L

0.0002% (2 g/mL)

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3.0 Preparation of E18 Rat CNS Cells for In Vitro Culture

3.1 Preparation of Primary Rat CNS Tissue and Initial Culture of Rat Neurospheres
1. Rat embryos (e.g. Sprague/Dawley or Fischer 344) are dissected at gestational day E18, where day E0 is the day a
gestational plug forms.
2. The brains are removed from the embryos and transferred to a 35 mm plate containing PBS plus 2% glucose, where routine
dissection procedures are then performed [Refer to Cell Biology: A Laboratory Handbook. ed. Julio E. Celis. 1998. Volume 1,
p149].
3. Dissect out cortex or other desired brain regions and place in PBS containing 2% glucose, on ice.
4. When dissections are complete, transfer tissue in PBS with 2% glucose into a 14 mL conical tube, allow tissues to settle and
pipette off supernatant.
5. Resuspend tissue in 1 mL "Complete" NeuroCult NS-A Proliferation Medium (Rat). Use a 1 mL pipettor with sterile plastic tip
to triturate the tissue approximately 5 - 10 times or until single cell suspension is achieved. To triturate, slightly tilt the tip and
press it against the bottom or side of the tube to generate resistance in order to break up the tissue.
Do not introduce air bubbles into the cell suspension. To maintain high viability, avoid using fire-polished glass pipettes to
disaggregate neurospheres derived from rat cells.
6. Add 1 mL "Complete" NeuroCult NS-A Proliferation Medium (Rat) to the single cell suspension and mix carefully to avoid
creating any bubbles. Leave for about 1 minute to allow the undispersed pieces of tissue to settle.
7. Transfer supernatant to a new sterile 14 mL tube. Discard undissociated tissue. Centrifuge supernatant at 150 x g (~800 rpm)
for 5 minutes. Discard supernatant.
8. Resuspend cells with a brief trituration (2 times) with a disposable plastic pipette tip in 1 mL "Complete" NeuroCult NS-A
Proliferation Medium (Rat).
9. Measure the exact volume and perform a viable cell count on a hemacytometer using a dilution (1/5 or 1/10, depending on
amount of tissue dissected) in Trypan Blue (Catalog #07050).
10.Seed cells at a density of 6 x 104 viable cells/mL or 1.2 x 105 viable cells/mL (see note in Section 4.0) in a T-25 cm2 tissue
culture flask (Nunc Catalog #156367 or VWR Catalog #15708-130). Use approximately 10 mL of "Complete" NeuroCult NS-A
Proliferation Medium (Rat). Incubate cultures in a 5% CO2 incubator at 37C. Refer to Figures 1 and 2 (Section 7.0) for photos
of rat neurospheres o1 and 2 days after plating.
Note: A partial medium change (25 - 30% of total volume) is highly recommended 2 or 3 days after plating, to
prevent the medium from becoming acidic. To change the medium, position the flask in an upright position and let
the cells and spheres settle to the bottom (2 - 3 minutes). Slowly remove ~3 mL of medium being careful not to
remove the cells, and replace the volume with fresh "Complete" NeuroCult NS-A Proliferation Medium (Rat).

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4
3.2 Preparation of Cells from Cryopreserved Neurospheres
3.2.1 Thawing Cryopreserved Rat Neurospheres
1. Warm Complete NeuroCult Proliferation Medium (Rat; made up as in Section 2.0) to 37C.
2. Add 9 mL of Complete NeuroCult Proliferation Medium (Rat) to a sterile 14 mL tube.
3. Remove the cryovial containing the cryopreserved E18 neurospheres from rat cortex (Catalog #00340) from the freezer and
thaw quickly in a 37C water bath.
4. Using a 1 mL disposable tip attached to a P1000 micropipettor, add 1 mL of Complete NeuroCult Proliferation Medium (Rat)
dropwise to the cryovial containing thawed neurospheres. Transfer the cell suspension to the tube prepared in step 2 and
centrifuge at 90 x g (~400 rpm) for 5 minutes.
5. Aspirate off all the supernatant. Add 10 mL of Complete NeuroCult Proliferation Medium (Rat) to the pellet and resuspend
neurospheres by pipetting gently (DO NOT DISSOCIATE NEUROSPHERES).
6. Transfer the entire cell suspension to a T-25 cm2 flask (Nunc Catalog #156367 or VWR Catalog #15708-130).
7. Observe the cell suspension under an inverted light microscope to determine appearance of neurospheres. The morphology of
the neurospheres may not look like intact spheres at this point due to the freeze/thaw process (the morphology should
recover the following day). This day (the day the neurospheres are thawed) is considered day 1. Culture in a humidified
incubator at 37C and 5% CO2.
8. Check cultures the next day and observe the morphology of the neurospheres. The neurospheres should mostly be floating
single neurospheres which look intact and viable (spheroid appearance) with only a minority of the neurospheres (<20%)
stuck down to the bottom of the flask. The neurospheres should be passaged at this point using the procedure described in
Section 3.2.2.

3.2.2 First Passage of Cryopreserved Rat Neurospheres

1. Pre-wet a 10 mL disposable pipette with Complete NeuroCult NS-A Proliferation Medium (Rat). Remove neurospheres and
medium and place in an appropriately-sized sterile tissue culture tube (e.g. 14 mL tube). Spin at 150 x g (~800 rpm) for 5
minutes. Remove supernatant, leaving behind approximately 150 - 180 L medium. Set the volume of a P200 pipettor with
sterile plastic tip and set the volume to slightly less than the approximate volume of the remaining medium (e.g. if the volume
of remaining medium is 180 L, set the volume of the pipettor to 160 L to avoid creating bubbles by expelling the entire cell
suspension volume). Pre-wet the tip with medium to reduce cells sticking inside the tip.
2. Gently triturate the cell pellet 10 - 15 times. Slightly tilt the tip and press it against the bottom or side of the tube to generate
resistance in order to break up the neurospheres. Rinse the side of the tube during trituration to remove the remaining
neurospheres that are attached to the side of the tube. If some neurospheres remain undissociated after 15 triturations (this
usually occurs at later passages), trituration can be extended to a maximum of 25 - 35 times.
To maintain high cell viability avoid using fire-polished glass pipettes to disaggregate neurospheres derived from rat cells.
3. Measure the volume of cells and medium. Count viable cells using Trypan Blue exclusion assay (1/10 dilution) on a
hemacytometer.
To avoid passaging cells as clumps, pipette cells up and down with a P200 pipette tip 4 - 8 times to obtain single cell
suspension prior to seeding cells in a flask.
4. Seed cells at densities of 1.25 x 104 and 2.5 x 104 viable cells/mL in a T-25 cm2 tissue culture flask (Nunc Catalog #156367 or
VWR Catalog #15708-130) containing 10 mL of "Complete" NeuroCult NS-A Proliferation Medium (Rat).
Two different seeding densities should be used to ensure that an appropriate number of cells are cultured for efficient
neurosphere formation.
5. Incubate cultures in a 5% CO2 incubator at 37C.
6. Cultures should be examined under the microscope regularly. A partial medium change should be performed 2 days after the
cultures are set up.
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4.0 Subculture of Rat Neurospheres

1. Observe the neurosphere cultures under a microscope to determine if the neurospheres are ready for passaging. Cultures of
rat E18 cortical or striatal neurospheres should be passaged every 3 - 4 days. Neurospheres should be passaged before the
diameter exceeds 100 m to avoid hypoxic cells in the centre of the spheres. Refer to Figure 3 (Section 7.0), for a photo of
neurospheres ready to be passaged.
Cells will proliferate as spheroids, called neurospheres, which in general detach from the surface of the tissue culture flask
and float in suspension. The neurospheres should be ready for subculture 3 - 4 days after plating depending on sphere
density and size. Viable neurospheres will, for the most part, be semi-transparent phase contrast bright, with many of the
cells on the outer surface displaying microspikes.
2. Remove neurospheres and medium and place in an appropriately-sized sterile tissue culture tube (e.g. 14 mL tube). Spin at
90 x g (~400 rpm), for 5 minutes. Remove supernatant, leaving behind approximately 150 - 180 L medium. Set the volume
of a P200 pipettor with sterile plastic tip to slightly less than the approximate volume of the remaining medium (e.g. if the
volume of remaining medium is 180 L, set the volume of the pipettor to 160 L to avoid creating bubbles by expelling the
entire cell suspension volume). Pre-wet the tip with medium to reduce cells sticking inside the tip.
3. Gently triturate the cell pellet 10 - 15 times. Slightly tilt the tip and press it against the bottom or side of the tube to generate
resistance in order to break up the neurospheres. Rinse the side of the tube during trituration to remove the remaining
neurospheres that are attached to the side of the tube. If some neurospheres remain undissociated after 15 triturations (this
usually occurs at later passages) trituration can be extended to a maximum of 25 - 35 times.
To maintain high cell viability, avoid using fire-polished glass pipette to disaggregate the neurospheres.
4. Measure the volume of cells and medium. Count viable cells using Trypan Blue exclusion assay (1/10 dilution at earlier
passages, lower dilution for later passages) on a hemacytometer.
To avoid passaging cells as clumps, pipette cells up and down with a P200 pipette tip 4 - 8 times to obtain single cell
suspension prior to seeding cells in a flask.
5. Seed cells at a density of 1.25 x 104 or 2.5 x 104 viable cells/mL (see note below) in a T-25 cm2 tissue culture flask (Nunc
Catalog #156367 or VWR Catalog #15708-130) containing 10 mL of "Complete" NeuroCult NS-A Proliferation Medium (Rat).
6. Incubate cultures in a 5% CO2 incubator at 37C.
7. Cultures should be examined under the microscope regularly. A partial medium change should be performed at Day 2 after
culture set-up as described in Section 3.1, Step 10.
Important Note:
Neural stem and progenitor cells from rat CNS tissue have different growth characteristics compared to mouse neural stem
and progenitor cells. The rate of proliferation of E18 rat cortical is higher (varies from 2 - >20 fold expansion of total cells
every 3 - 4 days of culture) during early passages (P2 to P5). A critical phase in the growth curve occurs between P5 and P9
(20 - 36 days) in culture, when a significant decrease in terms of fold expansion (average between 2 - 5 fold) is observed.
During this critical period, cells start to attach to the flask and differentiate. To reduce attachment of spheres to the flask, it is
important to perform partial medium changes every two days and be careful not to disrupt the attached cells so that only the
floating spheres are passaged. It is suggested that more than one flask is cultured during the critical passages, P5 - P9. Two
different seeding densities, 1.25 x 104 cells/mL and 2.5 x104 cells/mL, should be used to ensure a sufficient number of cells
are obtained for the next passage.

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Catalog #28725

5.0 Differentiation of Rat Neurospheres into Neurons, Astrocytes and

Oligodendrocytes
In the presence of rh EGF and rh FGF-b, rat neural stem cells and their progeny within neurospheres remain in a relatively
undifferentiated state. Upon removal of the growth factors and addition of a small amount of serum, differentiation to neurons,
astrocytes and oligodendrocytes is induced.

5.1 Preparation of NeuroCult NS-A Differentiation Medium (Rat)

Thaw one bottle containing 50 mL of NeuroCult NS-A Differentiation Supplements (Rat) at room temperature or overnight at 2 - 8C.
The NeuroCult NS-A Differentiation Supplements (Rat) can be aliquoted into 10 mL volumes and stored at -20C. However repeated
freezing and thawing is not recommended.
Add the entire volume (50 mL) of NeuroCult NS-A Differentiation Supplements (Rat) to the bottle (450 mL) of NeuroCult NS-A Basal
Medium (Rat) or add 1 mL of NeuroCult Differentiation Supplements (Rat) to every 9 mL of NeuroCult NS-A Basal Medium (Rat)
(1/10 dilution).
The "Complete" NeuroCult NS-A Differentiation Medium (Rat) is now ready for use.

5.2 Differentiation of EGF- and/or FGF-b-Responsive Rat Neurospheres

To initiate differentiation, single cells (dissociated from neurospheres) are cultured on commercially available 8-well culture slides (BD
BioCoat pre-coated with Poly-D-Lysine/Laminin Catalog #354688, or Poly-D-Lysine Catalog #354632) or on Poly-L-Ornithine coated
round glass coverslips (refer to Section 5.2.1).

5.2.1 Preparation of Poly-L-Ornithine Coated Glass Coverslips

1. If using round glass coverslips (Fisher Scientific #12-545-82) for immunolabeling, soak coverslips in 70% ethanol and
individually hand clean with a lint-free tissue. Sterilize ethanol-cleaned coverslips by autoclaving prior to coating. Coat
coverslips for 24 hours prior to use.
2. Using sterile forceps, transfer a single sterile glass coverslip to a well of a 24-well plate.
3. Dispense 1 mL of 15 g/mL Poly-L-Ornithine solution (Sigma Catalog #P3655) into each well.
4. Incubate glass coverslips for a minimum of 3 hours at 37C. After the incubation, remove the Poly-L-Ornithine solution from
each well by aspiration and rinse each well 3 x 15 minutes with sterile PBS (Catalog #37350).
5. The pre-coated Poly-L-Ornithine glass coverslips are now ready for the differentiation assay (Section 5.3).

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5.3 Differentiation Assay
1. If using the BD BioCoat 8-well culture slide (pre-coated with Poly-D-Lysine/Laminin BD Catalog #354688, or Poly-D-Lysine
Catalog #354632) add 0.75 mL/well of "Complete" NeuroCult NS-A Differentiation Medium (Rat). If using Poly-L-Ornithine
coated glass coverslips prepared as described in Section 5.2.1, place a single prepared coated coverslip into an individual
well of a 24-well culture dish (e.g. Corning Catalog #3526) containing 1 mL/well of "Complete" NeuroCult NS-A
Differentiation Medium (Rat).
2. After 3 - 4 days of culturing rat neurospheres in "Complete" NeuroCult NS-A Proliferation Medium (Rat), remove the medium
with suspended cells and place in an appropriate sized sterile tissue culture tube. Spin at 90 x g (~400 rpm) for 5 minutes.
3. Remove all of the supernatant and discard.
4. Resuspend the neurospheres in 150 - 180 L "Complete" NeuroCult NS-A Differentiation Medium (Rat). With a P200 pipette
tip, triturate the neurosphere suspension until single cell suspension is achieved (refer to Section 4, Steps 2 and 3 for a
description of trituration).
5. Resuspend the single cell suspension with 10 mL of Complete NeuroCult NS-A Differentiation Medium (Rat). Spin at
150 x g (~800 rpm) for 5 minutes. Remove all of the supernatant and discard.
6. Resuspend the cell pellet in approximately 200 L of Complete NeuroCult NS-A Differentiation Medium (Rat).
7. Measure the precise volume and count cell numbers using a dilution in Trypan Blue (1/5 or 1/10 dilution) and hemacytometer.
8. Prepare cells in an appropriate volume of Complete NeuroCult NS-A Differentiation Medium (Rat). Cells should be plated
with a density of 5 x 105 cells/mL on a coated-coverslip in a 24-well plate or at 1 x 105 cells/well in a BioCoat 8-well
chamber slide.
9. Observe cultures after 5 - 10 days with an inverted light microscope and determine if cells have differentiated and are viable.
10.Plates should be checked routinely to determine if the medium needs to be changed during the differentiation procedure. If
the medium becomes acidic (yellow), a half-medium change should be performed by removing approximately 50% of the
medium and replacing with fresh "Complete" NeuroCult NS-A Differentiation Medium (Rat).
11.Coverslips or pre-coated chamber slides containing differentiated rat neural cells can be removed after 5 - 10 days and
processed immediately for indirect immunofluorescence as described in Section 6.0.

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6.0 Immunolabeling Procedure

6.1 Fixation
1a.If using cells grown on pre-coated 8-well chamber slides, remove the culture medium from each chamber containing
differentiated cells (taking care not to remove all the medium to avoid exposure of the unfixed cells to air) and add 0.5 mL of
the 4% paraformaldehyde solution directly into the chamber. Incubate for 30 minutes at room temperature.
OR
1b.If using cells grown on coverslips, add 1 mL of 4% paraformaldehyde (in PBS pH 7.2) to a new 24-well plate. Transfer
coverslips containing differentiated cells into the paraformadehyde solution (one coverslip/well, cells facing up). Fix cells in
4% paraformaldehyde by incubation at room temperature for 30 minutes.
2. Aspirate the paraformaldehyde solution. For ease, an aspiration system connecting to a vacuum pump may be used.
3. Add PBS (pH 7.2) to the samples and incubate for 5 minutes. Repeat this washing procedure two more times for a total of 3
wash steps (aspiration with a vacuum pump is used each time to remove the supernatant).

6.2 Permeabilization
1. Permeabilize cells by adding 1 mL of PBS containing 0.3% Triton X-100 (Sigma Catalog #T9284) to each well and incubate for
5 - 10 minutes at room temperature.
2. Remove PBS/Triton-X 100 by aspiration. Perform 2 x 5 minute PBS washes.

6.3 Blocking and Labeling with Primary Antibodies

1. Make up a solution of PBS with 10% serum. This will be used as the diluent for the primary antibody. The type of serum used
depends on the secondary antibody chosen. Refer to Table 1 (below) to choose serum that is appropriate for different
secondary antibodies.
Table 1: Appropriate primary antibody blocking serum for different conjugated secondary antibodies

Secondary Antibody

Serum

Catalog #

Affini-Pure Goat anti-mouse IgM, chain specific FITC-conjugated

Affini-Pure Goat anti-rabbit IgG, (H+L) FITC-conjugated
Affini-Pure Goat anti-mouse IgG, (H+L) Texas Red dye-conjugated

goat
goat
goat

10211
10212
10213

Affini-Pure Goat anti-rabbit IgG, (H+L) AMCA-conjugated

goat

10214

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2. Dilute primary antibody in the appropriate serum-containing diluent (from Table 1). Appropriate working dilutions for
immunolabeling are shown in Table 2 (below).
Table 2: The optimal working dilution used for different primary antibodies

Targeted Antigen

Clone

Isotype

Catalog #

Working Dilution

GABA

rabbit polyclonal

01411

1:200

Glial Fibrillary Acidic Protein (GFAP)

rabbit polyclonal

01415

1:100

Microtubule Associated Protein-2 (MAP2)

AP20

mouse IgG1

01410

1:200

Myelin Basic Protein (MBP)

rabbit polyclonal

01417

1:200

Nestin

Rat 401

mouse IgG1

01418

1:50

TUJ1

mouse IgG2a

01409

1:1000

Oligodendrocyte Marker O4

O4

mouse IgM

01416

1:50

Oligodendrocyte Marker RIP

RIP

mouse IgG1

01433

1:100

Tyrosine Hydroxylase-2

TH2

mouse IgG1

01412

1:400

Neuronal Class III -Tubulin

The working dilutions are only recommendations as the optimal working dilution for each specific application should be
determined by the user.
3. Add diluted primary antibodies to the 24-well plate or chamber slides in a minimum volume of 250 L. Alternately place a
small volume of antibody, approximately 50 L, directly on the coverslip containing the differentiated cells and place a clean
second coverslip directly on top. Place in a hydrating chamber.
4. Incubate for 2 hours at 37C or overnight at 2 - 8C.
5. Wash off primary antibody with 3 x 5 minute washes using PBS.

6.4 Secondary Staining

1. Dilute secondary antibodies 1/100 in PBS + 2% serum (same serum used as diluent for the primary antibody, Table 1). Add a
minimum volume of 250 L to 24-well plate or chamber slide.
2. Incubate secondary antibodies for 30 minutes at 37C.
Note: Secondary antibody is sensitive to light and therefore keep samples in the dark whenever possible to prevent bleaching.
3. Wash off secondary antibody with 3 x 5 minute washes using PBS.
4. After the last wash add distilled water to each well.

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6.5 Mounting
If pre-coated chamber slides are used, perform Steps 1 - 2. If glass coverslips are used, then follow Steps 3 - 4.
1. If pre-coated 8-well chamber slides are used, follow manufacturer's protocol for removal of the chambers from the glass
slides. Rinse slides in distilled water in a coplin jar.
2. Add about 5 L of mounting medium (e.g. FluorSave Reagent, Calbiochem Catalog #345789) in each chamber slot and cover
with a 75 mm coverslip avoiding trapping any air bubbles.
3. If coverslips are used, add 10 L of mounting medium (e.g. FluorSave Reagent, Calbiochem Catalog #345789) to a clean
glass coverslip. Remove immunostained coverslip from the 24-well plate and gently tap corner of the coverslip to remove
excess water.
4. Place coverslip cell side down onto the mounting medium avoiding any air bubbles.
5. Visualize immunostaining under a fluorescent microscope using the appropriate filters for each fluorochrome (Table 3). Refer
to Section 8.0 for representative photographs of immunolabeled differentiated rat neurospheres.
Table 3: Peak wavelengths of absorption and emission for different fluorochrome-conjugated secondary antibodies

Fluorochrome

Absorption Peak (nm)

Emission Peak (nm)

Aminomethylcoumarin, AMCA
Fluorescein Isothiocyanate, FITC
Rhodamine Red-X, RRX

350
492
570

450
520
590

Texas Red, TR

596

620

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7.0 Representative Photographs of Cultured Rat Neurospheres

Figure 1. Small neurospheres, one day after plating

single cell suspensions of rat E18 cortex in Complete
NeuroCult NS-A Proliferation Media (Rat).
Magnification: 125X

Figure 2. Neurospheres derived from rat E18 cortical

cells after 2 days of culture in Complete NeuroCult
NS-A Proliferation Medium (Rat). The size (diameter) of
the neurospheres has increased.
Magnification: 125X

Figure 3. Neurospheres derived from rat E18 cortical

cells after 4 days in culture with Complete NeuroCult
NS-A Proliferation Medium (Rat). Neurospheres are
ready to be passaged. If not passaged, cells in the
center of the spheres will become dark and hypoxic and
the spheres will stick down to the bottom of flask.
Magnification: 125X

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Figure 4. The critical phase in rat neuropsheres

cultures: passages 5 - 9 (20 - 36 days in culture). At this
point some neurospheres will adhere to the flask and
start differentiating. It is important to perform a partial
medium change every other day and passage only the
floating neurospheres (to prevent differentiation).
Magnification: 125X

Figure 5. Neurospheres derived from rat E18 cortical

cells which have all adhered and differentiated after 3
days in culture. The medium should be regularly
replenished and only floating neurospheres passaged to
prevent cells attaching to the flask.
Magnification: 125X

Figure 6. Unhealthy neurospheres derived from rat

E18 cortical cells after 4 days in culture. Individual
neurospheres are beginning to dissociate into single
cells in culture, an indication of a problem with the
culture conditions or cells.
Magnification: 125X

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8.0 Representative Photographs of Immunolabeled Differentiated Rat Neurospheres

A

Figure 7. Differentiated rat neural cells cultured with NeuroCult NS-A Differentiation Medium (Rat). Cells are immunostained with lineage
specific antibodies as described in Section 6.3 and counterstained with DAPI (blue).
A. -Tubulin (red) and MBP (Green)
B. Oligodendrocyte Marker O4 (green) and -Tubulin (red)
C. Nestin (red)
D. GFAP (green) and MAP2 (red)
E and F. Triple staining with Oligodendrocyte Marker O4 (green), GFAP (blue) and -Tubulin (red)
Magnification: 400X
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MANUAL CATALOG #28725

VERSION 1.1.0