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 æl/cell to 96 well culture plates.
6. Incubate overnight at 37^oC.
7. Wash plates twice with PBS
8.  Add 125 æl/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.

 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

Procedure

1. Wash ELISA plates once with di-H₂O.
2. Add 250 æl/well PBS:2% BSA.
3.  Incubate 1 hour at 37^oC.
4. Wash 3 times with di-H₂O.
5.  Add 50 æl/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 æl/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 æl/well working substrate solution
    0.5 ml      4.0% OPD
    5 æl      30% H₂O₂
    1.0 ml      10X Substrate buffer
    8.5 ml      di-H₂O.
12. Incubate for 20 minutes at room temperature.
13. Add 25 æl/well 4.5N Sulfuric Acid
14. Read A₄₉₀

Notes

1.  Test all supernatants at 1:5 dilution.
2.  Test ascites at 1:100