细胞ELISA操作步骤医学心理学病毒学_高等教育-大学课件.pdf

Cellular ELISA Protocol Formalin Fixed Cell Plates 1.Tryps inize con flue nt flasks 2.Pool and count cells 3.Centrifuge at 1500 rpm for 10 minutes 4.Resuspe nd to the appropriate concen trati on in complete medium 4 x 10 5 cells/ml for epithelial cells 2 x 105 cells/ml for fibroblast cells 5.Add 100 卩 l/cell to 96 well culture plates.6.In cubate over ni ght at 37o C.7.Wash plates twice with PBS 8.Add 125 卩 l/well 10%Buffered Formalin 9.Fix for 15 minu tes at room temperature 10.Wash three times with di-H2O.11.Blot dry.12.Store at 2-8o C.Reage nts 1.PBS:1%BSA 2.PBS:2%BSA 3.Carbo nate Buffer 1.59 g Na2CO3 2.93 g NaHCO3 Dissolve in 900 ml di-H2O.Check pH and adjust to 9.6 necessary.Qs.to 1 liter.4.10X Substrate Buffer,pH 6.0 36.6 g Citric Acid,mon ohydrate 113.5 g Potassium dibasic phosphate Dissolve in 900 ml di-H2O.Check pH and adjust to 6.0 if n ecessary.Qs.to 1 liter.5.0.3%H2O2 Dilute 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 H2SO4 12.0 ml Concen trated Sulfuric Acid 88.0 ml di-H2O Procedure 1.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 in PBS: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 solution 0.5 ml 4.0%OPD H2O2 5 卩 l 30%1.0 ml 10X Substrate buffer 8.5 ml di-H20.12.I ncubate for 20 mi nu tes at room temperature.13.Add 25 卩 l/well 4.5N Sulfuric Acid 14.Read A490 Notes 1.Test all super nata nts at 1:5 dilutio n.Test ascites at 1:100