Tissue Homogenization Procedures for use with ELISA

Rat tissues

Procedure for preparing tissue homogenates made from rat skin:

1. Skin punches measuring 6 mm were homogenized in 1.5 mL extraction buffer (containing 10 mM
Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) per gram of tissue using a glass homogenizer. The
homogenates were transferred to 1.5 mL Eppendorf tubes, centrifuged at 13,000 xg for 10 minutes
at 4C, and the supernatant was stored at -80C until analyzed. TNF-alpha and IL-1beta protein
levels were determined using KRC3012 and KRC0012.

Reference: Blalock, T.D., J .C. Varela, S. Gowda, Y. Tang, C. Chen, B.A. Mast, and G.S. Schultz
(2001) Ischemic skin wound healing models in rats. Wounds 13(1):35-44.

2. Skin flap biopsies were obtained at different times after grafting and immediately stored under
frozen nitrogen. On the day of the assy, the biopsies were allows to thaw and 20-50 mg of wet
tissue was homgenized with the aid of a Polytron homogenizer in ice-cold PBS containing a
protease-inhibitor cocktail. The homogenates were centrifuged for 20 minutes at 10,000 x g to
remove debris and insoluble material, and aliquots of the supernatants were assayed for total
protein content by the BCA method and rat interferon-gamma was assayed by ELISA.

Reference: Fernandez-Botran, R., V. Gorantla, X.C. Sun, X.P. Ren, G. Perez-Abadia, F.A. Crespo,
R. Oliver, H.I. Orhu, E.E. Quan, C. Maldonado, M. Ray, and J .H. Barker (2002) Targeting of
glycosaminoglycan-cytokine interactions as a novel therapeutic approach in allotransplantation.
Transplantation 74(5):623-629 (cites the use of KRC4022 with skin biopsies).

Procedures for rat lung homogenization:

1. For Western blotting, lung tissue was homogenized with a tissue homogenizer in 5 volumes of
homogenization buffer (25 mM Tris-HCl, 2 mM EGTA, 1 mM benzamidine, 1 mM PMSF, pH 7.4).
Following centrifugation, (3,000 x g at 4 degrees C for 20 minutes, the supernatant was collected.
Total protein concentration was determined using Coomassie Plus Protein Assay. Samples (20
micrograms) were mixed with an equal volume of sample buffer (100 mM Tris-HCl, 2% SDS, 0.02%
bromophenol blue, and 10% glycerol and boiled for minutes then electrophoresed.
For ELISA, the lungs were homogenized in 5 volumes of buffer composed of 10 mM HEPES, 10
mM KCl, 0.5 M sucrose, 1 mM EGTA, 1 mM DTT. Homogenates were then centrifuged at 750 xg
for 10 minutes to isolate the nuclei. The supernatants which contained the cytosolic fraction were
stored at 70 degrees C and used for MIP-2 protein determination.

Reference: Calkins, C.M., D.D. Bensard, J .K. Heimbach, X. Meng, B.D. Shames, E.J . Pulido, R.C.
McIntyre (2001) L-arginine attenuates lipopolysaccharide induced lung chemokine production. Am.
J . Physiol. Lung Cell Mol. Physiol. 280(3):L400-408 (uses KRC1022 rat MIP-2 ELISA kit).

2. After BAL fluid collection, the left lungs were harvested for assessment of TNF-alpha, MIP-2, and
interferon-gamma protein expression. The lungs were homogenized using a tissue homogenizer
(Biospec Products, Racine, WI) in 1 mL lysis buffer containing 0.5% Triton X-100, 150 mM NaCl, 15
mM Tris, 1 mM CaCl, and 1 mM MgCl2, pH 7.4. Homogenates were then centrifuged at 10,000 x g
for 10 minutes. Supernatants were stored at 80 degrees C for later assessment.

Reference: Whitehead, G.S.; K.A. Grasman and E.C. Kimmel (2003) Lung function and airway
inflammation in rats following exposure to combustion products of carbon-graphite/epoxy composite
material: comparison to a rodent model of acute lung injury. Toxicology 183(1-3):175-197 (cites the
use KRC1022 with BALF and lung tissue homogenates).
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3. Frozen tissue samples were weighed and placed in homogenization buffer (4 degrees C) at a
ration of 100 mg tissue per milliliter, the buffer contained a protease-inhibitor combination including
1 mmol/L phenylmethylsulfonylfluoride, 1 microgram/mL peptstain A, 1 microgram/mL aproptinin,
and 1 microgram/mL leupeptin in phosphate buffered saline solution, pH 7.2, containing 0.05%
sodium azide and 0.5% Triton X-100. Samples were homogenized (Polytron Brinkman, Westbury
NY) and subjected to one round of freeze-thaw, sonicated for 10 minutes, and incubated at 4
degrees C for one hour. The final homogenate was centrifuged at 120,000 x g (ultracentrifuge).
Tissue supernatants were used for cytokine determination and these data are expressed per
milligram of tissue.

Reference: Phelan, H., P. Stahls, J . Hunt, G.J . Bagby, and P.E. Molina (2002) Impact of alcohol
intoxication on hemodynamic, metabolic, and cytokine responses to hemorrhagic shock. J . Trauma-
Injury Infection and Critical Care 52(4):675-682 (cites the use of KRC3012, KRC0812, KRC0062,
and KRC0102) with lung tissue homogenates and plasma samples).

4. Frozen tissues from the inferior and superior third of the lung were homogenized and incubated
at 4 degrees C in cell lysis buffer containing 10 mmol/L N-2-hydroxyethylpiperazine-N-2-
ethanesulfonic acid (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-
n,n-tetraacetic acid, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 0.6%
octylphenoxy-polyethoxy-ethanol (Nonidet P-40). Homogenates were then sonicated and then
centrifuged at 12,000 rpm for 10 minutes at 4 degrees C. The TNF-alpha, MIP-2, and IL-6 content
of the homogenates was determined with our ELISA kits. The total protein in the homogenates was
determined by the method of Bradford.

Reference: de Perrot, M., Y. Imai, G.A. Volgyesi, T.K. Waddell, M.Y. Liu, J .B. Mullen, K. McRae,
H.B. Zhang, A.S. Slutsky, V.M. Ranieri and S. Keshavjee (2002) Effect of ventilator-induced lung
injury on the development of reperfusion injury in a rat lung transplant. J ournal of Thoracic and
Cardiovascular Surgery 124(6):1137-1144 (cites the use of KRC0062, KRC3012, and KRC1022
with lung tissue homogenates).

Procedure for preparing cell lysates from pulmonary recruited and circulating PMNs

Lungs were surgically removed under anesthesia and lavaged with a total of 30 mL cold PBS
containing 0.1% dextrose using approximately 10 mL per lavage. Recovered lavage fluid was
centrifuged at 200 g for 5 minutes. The supernatant of lavage fluid collected in the first wash (7 mL
on average) was aliquoted and stored at 80 degrees C for chemokine determinations. The cell
pellet from each wash was suspended in PBS containing 0.1% dextrose and combined. The cell
counts were quantified under a light microscope with a hemacytometer. A cell monolayer was
prepared by cytocentrifugation and Wright-Giemsa stain was used to differentiate alveolar
macrophages and pulmonary recruited PMNs. Cells recovered from BAL fluid were subjected to
discontinuous Ficoll-Hypaque density gradient centrifugation to separate AMs and PMNs (as
previously described by Zhang, Bagby, et al., 1997). The isolated PMNs were washed twice with
PBS containing 0.1% dextrose.
Circulating PMNs were iolated from the whole blood samples by using NIM(TM )2 gradient
reagents based on the protocol supplied by the manufacturer. The viability of PMNs ilated from BAL
fluid and blood was over 95% as assessed by trypan blue exclusion. The purity of circulating PMNs
was greater than 90% as assessed by morphology using Wright-Giesma stain.

The isolated circulating and pulmonary recreuited PMNs were treated with a lysing buffer (PBS
containing 1% Triton X-100 and one tablet Complete Protease Inhibitor cocktail/7 mL lysing
solution). The cell lysates were kept at 80 degrees until analysis of the cell associated chemokine
by ELISA.

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References: Zhang, P., S. Nelson, M.C. Holmes, W.R. Summer and G.J . Bagby (2002)
Compartmentalization of macrophage inflammatory protein-2, but not cytokine-induced neutrophil
chemoattractant, in rats challenged with intratracheal endotoxin. Shock 17(2):104-108 (cites the
use of KRC1022 with plasma, BAL fluid, and cell lysates).

Zhang, P., G.J . Bagby, D.A. Stoltz, J .A. Spitzer, W.R. Summer, and S. Nelson (1997)
Modulation of the host response by granulocyte colony-stimulating factor in rats challenged with
intrapulmonary endotoxin. Shock 7:193-197.

Procedure for rat spleen and lung homogenizations

Procedure for the homogenization of rat spleen and lung for rat TNF-alpha determination:
Frozen tissue samples were weighed and homogenized (100 mg tissue per mL of homogenization
buffer). The homogenization buffer contained 1 mM PMSF, 1 microgram /mL pepstatin A, 1
microgram/mL aprotinin, and 1 microgram/mL leupeptin in phosphate buffered saline, pH 7.2, with
0.05% sodium azide and 0.5% Triton X 100. The samples were homogenized with a polytron and
then subjected to one round of freeze thaw, and then sonicated for 10 minutes, and then incubated
at 4 degrees C for 1 hour. The homogenate was centrifuged at 120,000 x g. The supernatants were
used for cytokine measurements. The data are expressed at pg/mg protein, with protein
concentration determined using the method of Lowry.

References: Molina, P.E. (2001) Opiate modulation of hemodynamic, hormonal, and cytokine
responses to hemorrhage. Shock 15(6): 471-478.
Molina, P.E. (2001) Noradrenergic inhibition of TNF upregulation in hemorrhagic
shock. Neuroimmunomodulation 9(3):125-133 (cites the use of KRC3012 in the measurement of
homogenates from lung and spleen tissue).

Procedure for rat fibroblasts

Subconfluent monolayers of sorted fibroblasts (based on whether they are Thy-1 positive or Thy-1
negative) were treated with IL-1beta orTNF-alpha and then lysed. Conditioned media were
collected in the presence of protease inhibitors centrifuged to remove debris, and concentrated 5
fold using a 3,000 MWCO filters (Centricon 3, Millipore). Cells were collected by scraping into cold
PBS with 2% FBS and protease inhibitors, and lysed by sonication, followed by centrifugation

Reference: Hagood, J .S., A. Mangalwadi, B.L. Guo, M.W. MacEwen, L. Salazar, and G.M. Fuller
(2002) Concordant and discordant interleukin-1-mediated signaling in lung fibroblast Thy-1
subpopulations. Am. J . Resp. Cell Mol. Biol. 26(6):702-708 (cites the use of KRC0812 with cell
lysates and cell culture supernatant).

Procedure for rat liver homogenizations

1. Procedure for the determination of rat TNF-alpha concentration in homogenates of rat livers:
Following administration of a lethal dose of LPS, the authors determined TNF-alpha concentrations
in the liver. Here is their homogenization procedure: Animals were killed and livers were rapidly
excised, rinsed of blood and homogenized by polytron (Brinkman) in homogenization buffer (PBS
containing 0.05% sodium azide, 0.5% Triton X-100, and a protease inhibitor cocktail, pH 7.2, 4C)
and then sonicated for 10 minutes. Homogenates were centrifuged at 12,000 x g for 10 minutes,
and TNF-alpha amounts in the supernatants were determined by ELISA. The TNF-alpha content
was expressed as pg/mg total protein. Total protein was determined by the Biorad assay.
Here is the lethal dose of LPS:15 mg/kg, given intravenously. They used E. coli 0111:B4 LPS 10
mg/mL in saline. The LPS solution was sonicated for 30 minutes prior to injection.

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Reference: Borovikova, L., S. Ivanova, M. Zhang, H. Yang, G.I. Botchkina, L.R. Watkins, H. Wang,
N. Abumrad, J .W. Eaton, and K.J . Tracey (2000) Vagus nerve stimulation attenuates the systemic
inflammatory response to endotoxin. Nature 405: 458-462 (cites the use of KRC3012 with liver
tissue homogenates).

2. Procedure for the homogenization of liver for rat IL-6 determination. The liver was sliced into
sections of about 500 mg on dry ice and them placed in 2 mL of ice cold phosphate buffered saline
containing 13 microliters/mL of protease inhibitor (Sigma catalog number P8340) plus 5% fetal calf
serum. The tissue was homogenized on ice and then was diluted to 5 mL with the same solution.
The homogenate was then centrifuged at 1500 xg for 15 minutes. The clarified supernatant was
then apportioned into 1 mL aliquots and stored at 70 degrees C until ready for use.

Reference: Lennie, T.A., M.D. Mortman, and R.J . Seeley (2001) Activity of body energy regulatory
pathways in inflammation-induced anorexia. Physiology and Behavior 73(4):517-523 (cites the use
of KRC0062 with serum and with tissue homogenates).

Procedure for rat liver and lung homogenization

1. Liver or lung samples (0.1 g) were homogenized in 1 mL ice-cold Tris lysis buffer (0.242% 2-
amino-2-hydroxymethyl-1,3-propanediol (TRIS) wt/wt in ddH2O with 5 mg/mL aprotinin, 5 mg/mL
leupeptin, 1 mg/mL pepstatin, and 10 mg/mL phenylmethylsulfonylfluoride dissolved in isopropanol)
and spun in a Beckman Allegra 6R centrifuge for 20 minutes at 2100 rpm.
Reference: J ho, D.H., T.A. Babcock, R. Tevar, W.S. Helton, and H.J . Espat (2002)
Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat
model of progressive non-metastasizing malignancy. J ournal of Parenteral and Enteral Nutrition
26(5):291-297 (cites the use of KRC1022 with lung and liver tissue homogenates).

Procedure of rat intestinal mucosa homogenates

Scrape the mucosa from the underlying muscle layer with a glass slide. The cells of the mucosa are
lysed with Tris EDTA (10 mM Tris-HCl, and 1 mM EDTA, pH 7.4) containing 0.05% sodium azide,
1% Tween-80, 2 mM PMSF, and 1 microgram per milliliter of each of the following protease
inhibitors: aprotinin, leupeptin, and pepstatin A. Homogenize the mucosa (20 s). Centrifuge the
homogenate (11,000 x g, 10 minutes at 4C). Collect the supernatant. Filter the supernatant (4.5
micron filter). Assay mucosal MIP-2 concentration with the rat MIP-2 ELISA kit (KRC1022) Express
the MIP-2 content as picograms MIP-2 per milligram of protein.

References: Castagliuolo, I., A.C. Keates, C.C. Wang, A, Pasha, L. Valenick, C.P. Kelly, S.T.
Nikulasson, J .T. LaMont, and C. Pothoulakis (1998) Clostridium difficile toxin A stimulates
macrophage-inflammatory protein-2 production in rat intestinal epithelial cells. J . Immunol.
160:6039-6045 (homogenates of rat intestinal mucosa).
Castagliulo, I., K. Karalis, L. Valenick, A. Pasha, S. Nikulasson, M. Wlk, and C.
Pothoulakis (2001) Endogenous corticosteroids modulate Clostridium difficile toxin A-induced
enteritis in rats. Am. J . Physiol. Gastrointest. Liver Physiol. 280:G539-545 (homogenates of rat
intestinal mucosa).

Procedure for rat spinal cord homogenizations

1. Procedure for IL-1 beta measurements in rat spinal cord homogenates: Spinal cord sample
weighed and then frozen at -70C. The spinal cord was homogenized in an ice bath with 9 mL of
cell culture medium (RPMI +10% heat inactivated fetal bovine serum) per gram of tissue using an
ultra-sound homogenizer. The homogenates were centrifuged (1310 x g) for 15 minutes at 4C. The
supernatant was used immediately for the IL-1 assay. Assay the at IL-1 beta concentration with
KRC0012, and express IL-1beta content as picograms per gram wet weight.
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Reference: Wang, C.X., J .A. Olschowka, and J .R. Wrathall (1997) Increase of IL-1beta mRNA and
protein in the spinal cord following experimental traumatic injury in the rat. Brain Research 759:190-
196.

2. IL-6 protein concentration was determined on L5 spinal cord from animals that were administered
systemic or central saline or systemic or central leflunomide therapy. Spinal cord homogenization
was peformed as follows: animlas were asphyxiated with CO2 and decaptitated. The spinal cord
was isolated at day 10 after surgery. Spinal cord was flast forzen on dry ice and stored at 80
degrees C until homogenization. At the time of homogenization, a 0.5 cm section of lunbar spinal
cord that included L4 and L5 level was removed from the intact frozen cord, weighed, minced and
placed in 0.25 mL of ice cold homongenization buffer containing protease inhibitors. Tissue was
homogenized with a Power Gen 125 tissue tearer set on high for 30 seconds. Samples were spun
at 20,000 x g for 30 minutes at 4 degrees C. The supernatant was taken and aliquoted and stored
at 80 degrees C until the time of assay.

References: Sweitzer, S.M. and J .A. DeLeo (2002) The active metabolite of leflunomide, an
immunosuppressive agent, reduces mechanical sensitivity in a rat mononeuropathy model. J ournal
of Pain 3(5):360-368 (cites the use of this kit with spinal cord homogenates).
Additional details are provided in the following reference:
Sweitser, S., D. Martin, J . DeLeo (2001) Intrathecal interleukin-1 receptor antagonist in
combination with soluble tumor necrosis factor receptor exhibit an anti-alppdynic action in a rat
model of neurophathic pain. Neuroscience 103:529-539.

Procedure for rat brain homogenizations

Procedure for IL-1beta, IL-10, MCP-1, MIP-2, and TNF-alpha in brain homogenates:
Cerebral cortex of each hemisphere was separately dissected and homogenized using Dounce
homogenizer in ice-cold lysis buffer containing HEPES 25 mM, pH 7.4, 3-[(3-cholamidopropyl)
dimethyl-ammonio]1-propanesulfonate 0.1%, MgCl2 5 mM, EDTA 1.3 mM, EGTA 1 mM, 10 g/mL
pepstatin, aprotinin, and leupeptin, and 1 mM PMSF. The homogenates were centrifuged (15
minutes at 50,000 rpm) and stored at -80C. Protein content was assayed by the bicinchoninic
(BCA) procedure. The cytokine content of the samples was assayed using KRC0012, KRC0102,
KRC1012, KRC1022, and KRC3012. The cytokine content was expressed at picogram cytokine
per milliliter per milligram.

Reference: Rabuffetti, M., C. Sciorati, G. Tarozzo, E. Clementi, A.A. Manfredi, and M. Beltramo
(2000) Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-
lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of
proinflammatory cytokines. J . Neurosci. 20(12):4398-4404 (cites the use of KRC0012, KRC3012,
KRC0102, KRC1012, and KRC1022 with brain homogenates).

Procedure for homogenizing rat fetal brain and placenta

Tissues were dissected and stored at -80C until ready for use. The tissues were placed in 1-30
volumes of 50 mM Tris-HCl buffer (pH 7.4) with 0.6 M NaCl, 0.2% Triton X-100, 1% BSA, 1 mM
benzamidine, 0.1 mM benzethonium chloride, and 0.1 mM PMSF. The tissues were homogenized
(PowerGen 125) on ice for 30 seconds and sonicated for 10 seconds at 10 mV. The homogenates
were centrifuged at 12.000 rpm for 20 minutes, and the supernatant were aliquoted and frozen at -
80C until assays were performed.
Reference: Urakubo, A., L.F. J arskog, J .A. Lieberman, and J .H. Gilmore (2001) Prenatal exposure
to maternal infection alters cytokine expression in the placenta, amniotic fluid, and fetal brain.
Schizophrenia Research 47:27-36.

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Procedure for spinal cord and eye homogenization

1. Freshly isolated spinal cords and whole eyes were homogenized in 1 mL sterile phosphate
buffered saline by sonication. The extracts were then clarified by centrifugation at 400 x g for 10
minutes. The collected supernatants were immediately frozen at 80 degrees C. A 50-100 microliter
of each sample was used in each test.

References: Adamus, G., M. Manczak, and M. Machnicki (2001) Expression of CC chemokines and
their receptors in the eye in autoimmune anterior uveitis associated with EAE. Investigative
Opthalmology and Visual Science 42(12):2894-2903 (cites the use of KRC1032 and KRC1012 with
tissue homogenates made from spinal cord and eye).
Manczak, M., S.G. J iang, B. Orzechowska, and G. Adamus (2002) Crucial role of
CCL3/MIP-1 alpha in the recurrence of autoimmune anterior uveitis induced with myelin basic
protein in Lewis rats. J . Autoimmunity 18(4):259-270 (cites the use of KRC0022, KRC0042,
KRC1012, and KRC1032 with tissue homogenates made by spinal cord and eye).

2. Corneas were asceptically removed and were then homogenized in 300 microliters of 50 mM
Tris, pH 7.4, containing 1 mM EDTA, 100 mM EDTA, 1 microtram of aportinin-isopropanaol per
milliliter, and transferred to a 1 mL tube and centrifuged at 7,000 x g for 20 minutes at 4 degrees C.
The cytokine content of the supernatant was determined with KRC0012 and KRC3012.

Reference: Saita, N., N. Fujiwara, I. Yano, K. Soejima, and K. Kobayashi (2000). Trehalose 6,6-
dimycolate (cord factor) of Mycobacterium tuberculosis induces corneal angiogenesis in rats. J .
Biol. Chem. 68(10):5991-5997 (cites the use of KRC3012 and KRC0012 with corneal tissue
homogenates).

Mouse tissues

Procedure for Mouse brain homogenization

The levels of cytokines were measured with brain homogenates made with saline or LPS treated
mice. Animals were sacrificed and decapitate 6 and 20 hours after injection. Appropriate brain
regions (hippocampus and Cortex) were dissected and snap frozen in 2-methylbutane on dry ice.
Brain samples were placed in sterile PBS containing a protease inhibitor cocktail, homogenized,
centrifuged at 11,000 rpm 20 minutes 4 degrees C and the supernatant removed. Protein
concentrations of all samples were measured using a BCA protein assay kit, and equivalent
amounts of proteins were used for the analyses. Cytokines levels were expressed as pg/mg.

Reference: Lee, J ., S.L. Chan, and M P. Mattson (2002) Adverse effect of a presenilin-1 mutation in
microglia results in enhanced nitric oxide and inflammatory cytokine responses to immune
challenge in the brain. Neuromolecular Medicine 2(1):29-45 (cites the use of KMC4022, KMC0012,
KMC3012, and KMC0062 kit with brain homogenates).

Procedure for the production of mouse periapical tissue homogenates

Frozen periapical tissue samples were ground using a precooled sterile mortar and pestle. The
tissue fragments were dispersed in 650 to 800 microliters of lysis buffer containing 100 microgram
of BSA fraction V, 100 micrograms Zwittergent-12, and 50 micrograms of gentamicin/mL, 10 mM
HEPES buffer, 1 microgram aprotinin and leupeptin/mL, 0.1 micromolar EDTA in RPMI 1640 as
described in Wang and Stashenk (1991). The incubation mixture was placed on ice and was
sonicated for 20-30 seconds. The sonication material was centrifuged and the supernatant was
collected and stored frozen at 70 degrees C until the ELISAs were performed.

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References: Balto, K., H. Sasaki, and P. Stashenko (2001) Interleukin-6 deficiency increases
inflammatory bone destruction. Infection and Immunity February:744-750 (cites the use of
KMC0022, KMC0044, KMC0062, KMC0012, KMC4022, and KMC3012 with periapical tissue
homogenates).
Wang, C.Y. and P. Stashenk (1991) The role of interleukin-1 alpha in the pathogenesis
of periapical bone destruction in a rat model system. Oral Microbiol. Immunol. 8:50-56.

Procedure for preparation of mouse splenic tumor and cytosolic fractions

1. Splenic tumor tissue was excised, immediately frozen in liquid N2 and stored at -70C. frozen
tissues were thawed in ice-cold homogenization buffer containing 50 mM potassium phosphate, pH
7.1, 0.1 M NaCl, 2 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride, 60 micrograms/mL soybean
trypsin inhibitor, 2 micrograms/mL leupeptin, 2 micrograms/mL aprotinin, 2 micrograms/mL
pepstatine. Tissues were disrupted twice, for 10 seconds each, on ice using a tissue tearer from
IKA Labortechnik. The samples were homogenized by sonication at 4 degreees C using a Cole
Parmer 4710 ultrasonic homogenizer. Debris was removed by centrifugation at 100,000 x g for 15
minutes at 4 degrees C, and the resultant supernatant was centrifuged at 100,000 x g for 30
minutes at 4 degrees C. The supernatant contains the cytosolic protein and the pellet contains the
membrane fraction.

Reference: Yao, M., S, Kargman, E.C. Lam, C.R. Kelly, Y.X. Zheng, P. Luk, E. Kwong, J .F. Evans
and M.M. Wolfe (2003) Inhibition of cyclooxygenase-2 by rofecoxib attenuates the growth and
metastatic potential of colorectal carcinoma in mice. Cancer Research 63(3):586-592 (cites the use
of this kit with splenic tumor cytosolic fractions).

Human tissues

Preparation of human tissue homogenates

1. Brain Sonication protocol for IL-1 beta ELISA (KHC0012)
developed by Kien T. Nguyen (11-6-97).

Extraction Buffer: (Store at 4C for up to 2 weeks)

Iscove's Medium (190 mL/200 mL total volume) Gibco #12440-020
Fetal Calf Serum (10 mL/200 mL total volume) Gibco #16000-044


10X Enzyme Cocktail: (Store at 4C for up to 2 weeks)

Conc. Chemical Amount Supplier/Catalog #
100mM Amino-n-caproic acid (FW 131.2) 13.10 g Sigma #A-7824
10mM EDTA (FW 372.2) 3.722g Sigma #E-5134
5mM Benzamidine (FW 156.6) 0.783g Sigma #B-6506
Dissolve the above in 99 mL d.d. water.


PMSF:

0.2 mM Phenylmethyl sulfonyl fluoride 0.174 g Sigma #P-7626
(FW 174.2)
Dissolve PMSF in 5 mL isopropanol. (Vortex vigorously, may not all go into solution)


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Prior to weighing the tissues, prepare Sonication Buffer and add 0.5-1.0 mL to each tube,
depending on tissue weight.

Sonication Buffer:

For 15 mL, Add:
13.5 mL Extraction Buffer
1.485 mL Enzyme Cocktail
15 L PMSF

PMSF is very unstable, thus sonication buffer should be made fresh prior to sonication of each set
of tissues.

Add each tissue to sonication buffer sitting in ice water, each sample is sonicated for 10 seconds at
setting 10 using a Microson Ultrasonic Cell Disrupter (Heat Systems Ultrasonic, Inc. model #MS-
50). Sonicated supernatant is removed to a clean 1.5 mL snap-cap Eppendorf tube and stored on
ice water until centrifugation. Samples are centrifuged at 4C, 10,000 rpm, 10 minutes on an
Eppendorf Centrifuge, model #5403. Once completed, clean supernatants are transferred to a
second clean 1.5 mL snap-cap Eppendorf tube and stored at 4C until the ELISA is performed.

2. Yamaoka et al. cite the use of KHC0102 with tissue homogenates made from biopsy specimens
taken from the antrum under endoscopy. Five biopsy specimens were taken from the antrum, using
the same size forceps from similar cites at each endoscopy. Two were used for cytokine
measurement. The biopsies were placed in 2.0 mL PBS, pH 7.4, and immediately frozen on dry
ice, and stored at -80C until use. Samples were homogenized using a tissue homogenizer
(Kontes), centrifuged for 10 minutes at 10,000 rpm, then assayed for total protein content using a
modified Lowry method. The supernatants were diluted to a concentration of 0.5 mg/mL and were
stored at -80C until ready for analysis. Data were expressed as pg IL-10/mg protein.

Reference: Yamaoka, Y., M. Kita, T. Kodama, N. Sawai, K. Kashima, and J . Imanishi (1997)
Induction of various cytokines and development of severe mucosal inflammation by cagA gene
positive Helicobacter pylori strains. Gut 41:442-451.

3. MRC5 cells were plated in 25 cm
2
flasks and allowed to grow to confluence. The cells were
washed twice in sterile PBS. Four mL of serum-free medium was replaced. At specific times,
supernatants were harvested and cells were washed twice with PBS and scraped. The cells were
pelleted and lysed in 10 microliters lysis solution,containing 500 mM Tris, pH 7.4, 0.01% Triton X-
100, 1 mM PMSF, and 5 micrograms/mL aporotinin, pepstatin A, and leupeptin. Supernatants and
cells were stored at 80 degrees C until analysis. To analyze the amount of RANTES in the cell
lysate, the authors ran a standard curve which was made up in the same buffer as the cell lysate (ie
contained Triton X-100).

Reference: Randolph-Habecker, J ., B. Rahill, B. Torok-Storb, J . Vieira, P. E. Kolattukudy, B. H.
Rovin, and D. D. Sedmak (2002) The expression of the cytomegalovirus chemokine receptor
homolog US28 sequesters biologically active CC chemokines and alters IL-8 production. Cytokine
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