metode isolasi jaringan utk elisa (invitrogen)

8/11/2019 Metode Isolasi Jaringan Utk ELISA (Invitrogen)

1/8

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 mMTris pH 7.4, 150 mM NaCl, 1% Triton X-100) per gram of tissue using a glass homogenizer. Thehomogenates 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 proteinlevels were determined using KRC3012and 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 underfrozen nitrogen. On the day of the assy, the biopsies were allows to thaw and 20-50 mg of wettissue 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 toremove debris and insoluble material, and aliquots of the supernatants were assayed for totalprotein 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 ofglycosaminoglycan-cytokine interactions as a novel therapeutic approach in allotransplantation.Transplantation 74(5):623-629 (cites the use of KRC4022with skin biopsies).

Procedures for rat lung homogenization:

1. For Western blotting, lung tissue was homogenized with a tissue homogenizer in 5 volumes ofhomogenization 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 (20micrograms) 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, 10mM KCl, 0.5 M sucrose, 1 mM EGTA, 1 mM DTT. Homogenates were then centrifuged at 750 xgfor 10 minutes to isolate the nuclei. The supernatants which contained the cytosolic fraction werestored 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 KRC1022rat 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, 15mM Tris, 1 mM CaCl, and 1 mM MgCl2, pH 7.4. Homogenates were then centrifuged at 10,000 x gfor 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 airwayinflammation in rats following exposure to combustion products of carbon-graphite/epoxy compositematerial: comparison to a rodent model of acute lung injury. Toxicology 183(1-3):175-197 (cites theuse KRC1022with BALF and lung tissue homogenates).

www.invitrogen.com

Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 920081


2/8

3. Frozen tissue samples were weighed and placed in homogenization buffer (4 degrees C) at aration of 100 mg tissue per mill iliter, the buffer contained a protease-inhibitor combination including1 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, WestburyNY) and subjected to one round of freeze-thaw, sonicated for 10 minutes, and incubated at 4degrees 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 permilligram of tissue.

Reference:Phelan, H., P. Stahls, J. Hunt, G.J. Bagby, and P.E. Molina (2002) Impact of alcoholintoxication 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 incubatedat 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 thencentrifuged at 12,000 rpm for 10 minutes at 4 degrees C. The TNF-alpha, MIP-2, and IL-6 contentof the homogenates was determined with our ELISA kits. The total protein in the homogenates wasdetermined 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 lunginjury on the development of reperfusion injury in a rat lung transplant. Journal of Thoracic andCardiovascular Surgery 124(6):1137-1144 (cites the use of KRC0062, KRC3012, and KRC1022with 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 PBScontaining 0.1% dextrose using approximately 10 mL per lavage. Recovered lavage fluid wascentrifuged at 200 g for 5 minutes. The supernatant of lavage fluid collected in the first wash (7 mLon average) was aliquoted and stored at 80 degrees C for chemokine determinations. The cellpellet from each wash was suspended in PBS containing 0.1% dextrose and combined. The cellcounts were quantified under a light microscope with a hemacytometer. A cell monolayer wasprepared by cytocentrifugation and Wright-Giemsa stain was used to differentiate alveolarmacrophages and pulmonary recruited PMNs. Cells recovered from BAL fluid were subjected todiscontinuous Ficoll-Hypaque density gradient centrifugation to separate AMs and PMNs (aspreviously described by Zhang, Bagby, et al., 1997). The isolated PMNs were washed twice withPBS 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 BALfluid and blood was over 95% as assessed by trypan blue exclusion. The purity of circulating PMNswas greater than 90% as assessed by morphology using Wright-Giesma stain.

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

www.invitrogen.com



3/8

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 neutrophilchemoattractant, in rats challenged with intratracheal endotoxin. Shock 17(2):104-108 (cites theuse of KRC1022with 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 withintrapulmonary 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 homogenizationbuffer). The homogenization buffer contained 1 mM PMSF, 1 microgram /mL pepstatin A, 1microgram/mL aprotinin, and 1 microgram/mL leupeptin in phosphate buffered saline, pH 7.2, with0.05% sodium azide and 0.5% Triton X 100. The samples were homogenized with a polytron andthen subjected to one round of freeze thaw, and then sonicated for 10 minutes, and then incubatedat 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 proteinconcentration determined using the method of Lowry.

References:Molina, P.E. (2001) Opiate modulation of hemodynamic, hormonal, and cytokineresponses to hemorrhage. Shock 15(6): 471-478.

Molina, P.E. (2001) Noradrenergic inhibition of TNF upregulation in hemorrhagicshock. Neuroimmunomodulation 9(3):125-133 (cites the use of KRC3012in the measurement ofhomogenates from lung and spleen tissue).

Procedure for rat fibroblasts

Subconfluent monolayers of sorted fibroblasts (based on whether they are Thy-1 positive or Thy-1negative) were treated with IL-1beta orTNF-alpha and then lysed. Conditioned media werecollected in the presence of protease inhibitors centrifuged to remove debris, and concentrated 5fold using a 3,000 MWCO filters (Centricon 3, Millipore). Cells were collected by scraping into coldPBS 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-1subpopulations. Am. J. Resp. Cell Mol. Biol. 26(6):702-708 (cites the use of KRC0812with celllysates 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 concentrationsin 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 (PBScontaining 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 contentwas 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. coli0111:B4 LPS 10mg/mL in saline. The LPS solution was sonicated for 30 minutes prior to injection.

www.invitrogen.com



4/8

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 systemicinflammatory response to endotoxin. Nature 405: 458-462 (cites the use of KRC3012with livertissue homogenates).

2. Procedure for the homogenization of liver for rat IL-6 determination. The liver was sliced intosections of about 500 mg on dry ice and them placed in 2 mL of ice cold phosphate buffered salinecontaining 13 microliters/mL of protease inhibitor (Sigma catalog number P8340) plus 5% fetal calfserum. 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 wasthen 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 regulatorypathways in inflammation-induced anorexia. Physiology and Behavior 73(4):517-523 (cites the useof KRC0062with 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/mLleupeptin, 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 ratmodel of progressive non-metastasizing malignancy. Journal of Parenteral and Enteral Nutrition26(5):291-297 (cites the use of KRC1022with 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 arelysed 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 proteaseinhibitors: 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 (4.5micron filter). Assay mucosal MIP-2 concentration with the rat MIP-2 ELISA kit (KRC1022) Expressthe 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 stimulatesmacrophage-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-inducedenteritis in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 280:G539-545 (homogenates of ratintestinal 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 ofcell 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-1assay. Assay the at IL-1 beta concentration withKRC0012, and express IL-1beta content as picograms per gram wet weight.

www.invitrogen.com



5/8

Reference:Wang, C.X., J.A. Olschowka, and J.R. Wrathall (1997) Increase of IL-1beta mRNA andprotein 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 administeredsystemic or central saline or systemic or central leflunomide therapy. Spinal cord homogenizationwas peformed as follows: animlas were asphyxiated with CO2 and decaptitated. The spinal cordwas isolated at day 10 after surgery. Spinal cord was flast forzen on dry ice and stored at 80degrees C until homogenization. At the time of homogenization, a 0.5 cm section of lunbar spinalcord that included L4 and L5 level was removed from the intact frozen cord, weighed, minced andplaced in 0.25 mL of ice cold homongenization buffer containing protease inhibitors. Tissue washomogenized with a Power Gen 125 tissue tearer set on high for 30 seconds. Samples were spunat 20,000 x g for 30 minutes at 4 degrees C. The supernatant was taken and aliquoted and storedat 80 degrees C until the time of assay.

References:Sweitzer, S.M. and J.A. DeLeo (2002) The active metabolite of leflunomide, animmunosuppressive 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 incombination with soluble tumor necrosis factor receptor exhibit an anti-alppdynic action in a ratmodel 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 Douncehomogenizer 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/mLpepstatin, 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 cytokineper 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 ofproinflammatory cytokines. J. Neurosci. 20(12):4398-4404 (cites the use of KRC0012, KRC3012,KRC0102, KRC1012, and KRC1022with 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 1-30volumes of 50 mM Tris-HCl buffer (pH 7.4) with 0.6 M NaCl, 0.2% Triton X-100, 1% BSA, 1 mMbenzamidine, 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 homogenateswere 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. Jarskog, J.A. Lieberman, and J.H. Gilmore (2001) Prenatal exposureto maternal infection alters cytokine expression in the placenta, amniotic fluid, and fetal brain.Schizophrenia Research 47:27-36.

www.invitrogen.com



6/8

Procedure for spinal cord and eye homogenization

1. Freshly isolated spinal cords and whole eyes were homogenized in 1 mL sterile phosphatebuffered saline by sonication. The extracts were then clarified by centrifugation at 400 x g for 10minutes. 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 andtheir receptors in the eye in autoimmune anterior uveitis associated with EAE. InvestigativeOpthalmology and Visual Science 42(12):2894-2903 (cites the use of KRC1032 and KRC1012 withtissue homogenates made from spinal cord and eye).

Manczak, M., S.G. Jiang, B. Orzechowska, and G. Adamus (2002) Crucial role ofCCL3/MIP-1 alpha in the recurrence of autoimmune anterior uveitis induced with myelin basicprotein in Lewis rats. J. Autoimmunity 18(4):259-270 (cites the use of KRC0022, KRC0042,KRC1012, and KRC1032with tissue homogenates made by spinal cord and eye).

2. Corneas were asceptically removed and were then homogenized in 300 microliters of 50 mMTris, 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 KRC0012and KRC3012.

Reference:Saita, N., N. Fujiwara, I. Yano, K. Soejima, and K. Kobayashi (2000). Trehalose 6,6-dimycolate (cord factor) ofMycobacterium tuberculosisinduces corneal angiogenesis in rats. J.Biol. Chem. 68(10):5991-5997 (cites the use of KRC3012and KRC0012with corneal tissuehomogenates).

Mouse tissues

Procedure for Mouse brain homogenization

The levels of cytokines were measured with brain homogenates made with saline or LPS treatedmice. Animals were sacrificed and decapitate 6 and 20 hours after injection. Appropriate brainregions (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. Proteinconcentrations of all samples were measured using a BCA protein assay kit, and equivalentamounts 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 inmicroglia results in enhanced nitric oxide and inflammatory cytokine responses to immunechallenge in the brain. Neuromolecular Medicine 2(1):29-45 (cites the use of KMC4022, KMC0012,KMC3012, and KMC0062kit 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. Thetissue fragments were dispersed in 650 to 800 microliters of lysis buffer containing 100 microgramof BSA fraction V, 100 micrograms Zwittergent-12, and 50 micrograms of gentamicin/mL, 10 mMHEPES buffer, 1 microgram aprotinin and leupeptin/mL, 0.1 micromolar EDTA in RPMI 1640 asdescribed in Wang and Stashenk (1991). The incubation mixture was placed on ice and wassonicated for 20-30 seconds. The sonication material was centrifuged and the supernatant wascollected and stored frozen at 70 degrees C until the ELISAs were performed.

www.invitrogen.com



7/8


8/8

www.invitrogen.com


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 Buffer1.485 mL Enzyme Cocktail

15 L PMSF

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

Add each tissue to sonication buffer sitting in ice water, each sample is sonicated for 10 seconds atsetting 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 anEppendorf 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 KHC0102with tissue homogenates made from biopsy specimenstaken from the antrum under endoscopy. Five biopsy specimens were taken from the antrum, usingthe same size forceps from similar cites at each endoscopy. Two were used for cytokinemeasurement. 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 amodified 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 pyloristrains. Gut 41:442-451.

3. MRC5 cells were plated in 25 cm2flasks 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 werepelleted 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 andcells were stored at 80 degrees C until analysis. To analyze the amount of RANTES in the celllysate, the authors ran a standard curve which was made up in the same buffer as the cell lysate (iecontained 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 receptorhomolog US28 sequesters biologically active CC chemokines and alters IL-8 production. Cytokine19(1):37-46 (cites the use of the human RANTES ELISA kit KHC1032with cell culture supernatantsamples and with cell lysates).

metode isolasi jaringan utk elisa (invitrogen)

Documents