Cellular ELISA ProtocolFormalin Fixed Cell Plates1. Tryps inize con flue nt flasks2. Pool and count cells3. Centrifuge at 1500 rpm for 10 minutes4. Resuspe nd to the appropriate concen trati on in complete medium4 x 105 cells/ml for epithelial cells2 x 105 cells/ml for fibroblast cells5. Add 100 卩 l/cell to 96 well culture plates.6. In cubate over ni ght at 37o C.7. Wash plates twice with PBS8. Add 125 卩 l/well 10% Buffered Formalin9. Fix for 15 minu tes at room temperature10. Wash three times with di-H2O.11. Blot dry.12. Store at 2-8o C.Reage nts1. PBS:1% BSA2. PBS:2% BSA3. Carbo nate Buffer1.59 g Na2CO32.93 g NaHCO3Dissolve in 900 ml di-H2O. Check pH and adjust to 9.6necessary. Qs. to 1 liter.4. 10X Substrate Buffer, pH 6.036.6 g Citric Acid, mon ohydrate113.5 g Potassium dibasic phosphateDissolve in 900 ml di-H2O. Check pH and adjust to 6.0 if n ecessary. Qs. to 1 liter.5. 0.3% H2O2Dilute 30% stock Peroxide 1:100 in di-H2O.6. OPD Stock, 4.0%4 g OPD in 100 ml di-H2O. Aliquot and store at -20o C.Protect from light.4.5N H2SO412.0 ml Concen trated Sulfuric Acid88.0 ml di-H2OProcedure1. Wash ELISA plates once with di-H2O.2. Add 250 卩 l/well PBS:2% BSA.3. Incubate 1 hour at 37o C.4. Wash 3 times with di-H2O.5. Add 50 卩 l/well supe, ascites;ontrols diluted inPBS:1%BSA.6. I ncubate for 2 hr at 37o C.7. Wash 5 times with di-H2O.8. Add 50 卩 l/well antouse lgG:HRP diluted in PBS:1% BSA.9. I ncubate for 1 hr at 37o C.10. Wash 5 times with di-H2O. Wash on ce with carbo nate buffer.11. Add 50 卩 l/well working substrate solution0.5 ml 4.0% OPDH2O25 卩 l 30% 1.0 ml 10X Substrate buffer8.5 ml di-H20.12. I ncubate for 20 mi nu tes at room temperature.13. Add 25 卩 l/well 4.5N Sulfuric Acid14. Read A490Notes1. Test all super nata nts at 1:5 dilutio n.Test ascites at 1:100