| TissueHomogenizationProcedures 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 4°C, and the supernatant was stored at -80°C 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 immediay 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）. 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. Journal 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 PMN’s Lungs were surgically removed under anesthesia and lavaged with a total of 30 mL cold PBS containing 0.1% dextrose using approximay 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. 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, 4˚C） 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. 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: Jho, 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. Journal 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 4°C）. 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 -70°C. 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 4°C. The supernatant was used immediay for the IL-1β assay. Assay the at IL-1 beta concentration with KRC0012, and express IL-1beta content as picograms per gram wet weight. 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. Journal 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 separay 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 μg/mL pepstatin, aprotinin, and leupeptin, and 1 mM PMSF. The homogenates were centrifuged （15 minutes at 50,000 rpm） and stored at -80°C. 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 -80°C 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 -80°C until assays were performed. Reference: Urakubo, A., L.F. Jarskog, 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. 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 immediay 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. Jiang, 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 ELISA’s were performed. 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, immediay frozen in liquid N2 and stored at -70°C. 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 4˚C 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 4°C for up to 2 weeks）
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） 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 μL 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 4°C, 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 4°C 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 immediay frozen on dry ice, and stored at -80°C 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 -80°C 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 cm2 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 19（1）:37-46 （cites the use of the human RANTES ELISA kit KHC1032 with cell culture supernatant samples and with cell lysates）. | ||||||||||||||||
