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 m l/cell to 96 well culture plates.

6. Incubate overnight at 37o C.

7. Wash plates twice with PBS

8. Add 125 m l/well 10% Buffered Formalin

9. Fix for 15 minutes at room temperature

10. Wash three times with di-H2 O.

11. Blot dry.

12. Store at 2-8o C.

B. Reagents

1. PBS:1% BSA

2. PBS:2% BSA

3. Carbonate Buffer 1.59 g Na2 CO3 2.93 g NaHCO3 Dissolve in 900 ml di-H2 O. 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-H2 O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter.

5. 0.3% H2 O2 Dilute 30% stock Peroxide 1:100 in di-H2 O.

6. OPD Stock, 4.0% 4 g OPD in 100 ml di-H2 O. Aliquot and store at -20o C. Protect from light.

7. 4.5N H2 SO4 12.0 ml Concentrated Sulfuric Acid 88.0 ml di-H2 O

B. Procedure

1. Wash ELISA plates once with di-H2 O.

2. Add 250 m l/well PBS:2% BSA.

3. Incubate 1 hour at 37o C.

4. Wash 3 times with di-H2 O.

5. Add 50 m l/well supe, ascites, or controls diluted in PBS:1%BSA.

6. Incubate for 2 hr at 37o C.

7. Wash 5 times with di-H2 O.

8. Add 50 m l/well anti-mouse IgG:HRP diluted in PBS:1% BSA.

9. Incubate for 1 hr at 37o C.

10. Wash 5 times with di-H2 O. Wash once with carbonate buffer.

11. Add 50 m l/well working substrate solution 0.5 ml 4.0% OPD 5 m l 30% H2 O2 1.0 ml 10X Substrate buffer 8.5 ml di-H2 O.

12. Incubate for 20 minutes at room temperature.

13. Add 25 m l/well 4.5N Sulfuric Acid

14. Read A490

C. Notes

1. Test all supernatants at 1:5 dilution.

2. Test ascites at 1:100

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