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 ml/cell to 96 well culture plates.

6.     Incubate overnight at 37^oC.

7.     Wash plates twice with PBS

8.     Add 125 ml/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 2-8^oC.

B.  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 -20^oC.  Protect from light.

7.     4.5N H₂SO₄
12.0 ml      Concentrated Sulfuric Acid
88.0 ml      di-H₂O

B.    Procedure

1.     Wash ELISA plates once with di-H₂O.

2.     Add 250 ml/well PBS:2% BSA.

3.     Incubate 1 hour at 37^oC.

4.     Wash 3 times with di-H₂O.

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

6.     Incubate for 2 hr at 37^oC.

7.     Wash 5 times with di-H₂O.

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

9.     Incubate for 1 hr at 37^oC.

10. Wash 5 times with di-H₂O.  Wash once with carbonate buffer.

11. Add 50 ml/well working substrate solution
0.5 ml      4.0% OPD
5 ml      30% H₂O₂
1.0 ml      10X Substrate buffer
8.5 ml      di-H₂O.

12. Incubate for 20 minutes at room temperature.

13. Add 25 ml/well 4.5N Sulfuric Acid

14. Read A₄₉₀

C.    Notes

1.     Test all supernatants at 1:5 dilution.

2.     Test ascites at 1:100