Cellular ELISA Protocol
Formalin Fixed Cell Plates
1. Trypsinize confluent flasks
2. Pool and count cells
3. Centrifuge at 1500 rpm for 10 minutes
4. Resuspend to the appropriate concentration in complete medium 4 x 105 cells/ml for epithelial cells 2 x 105 cells/ml for fibroblast cells
5. Add 100 μl/cell to 96 well culture plates.
6. Incubate overnight at 37oC.
7. Wash plates twice with PBS
8. Add 125 μl/well 10% Buffered Formalin
9. Fix for 15 minutes at room temperature
10. Wash three times with di-H2O.
11. Blot dry.
12. Store at 2-8oC.
Reagents
1. PBS:1% BSA
2. PBS:2% BSA
3. Carbonate 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, monohydrate 113.5 g Potassium dibasic phosphate Dissolve in 900 ml di-H2O. Check pH and adjust to 6.0 if necessary. 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 -20oC. Protect from light.
4.5N H2SO4 12.0 ml Concentrated Sulfuric Acid 88.0 ml di-H2O
Procedure
1. Wash ELISA plates once with di-H2O.
2. Add 250 μl/well PBS:2% BSA.
3. Incubate 1 hour at 37oC.
4. Wash 3 times with di-H2O.
5. Add 50 μl/well supe, ascites, or controls diluted in PBS:1%BSA.
6. Incubate for 2 hr at 37oC.
7. Wash 5 times with di-H2O.
8. Add 50 μl/well anti-mouse IgG:HRP diluted in PBS:1% BSA.
9. Incubate for 1 hr at 37oC.
10. Wash 5 times with di-H2O. Wash once with carbonate buffer.
11. Add 50 μl/well working substrate solution 0.5 ml 4.0% OPD 5 μl 30% H2O2 1.0 ml 10X Substrate buffer 8.5 ml di-H2O.
12. Incubate for 20 minutes at room temperature.
13. Add 25 μl/well 4.5N Sulfuric Acid
14. Read A490
Notes
1. Test all supernatants at 1:5 dilution.
Test ascites at 1:100
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