|
1. Cell Plating-no Test Reagent/Drug |
Seed cells at 1-2 x 105 cells/ml, 100 ml/well |
| 2. Cell Plating-withTest Reagent/Drug (see below step 3) |
Seed cells at 0.5-4 x 105 cells/ml, 50 ml/well |
| 3. Addition of Test Reagent(s)/Drug | Add 50 ml/well, 2X concentration desired |
| 4. Addition of BrdU | Dilute 500X stock BrdU, add 20 ml/well (be sure to include a No BrdU control) |
| 5. Incubate | 2-24 hours |
| 6. Fix and Denature -Adherent Cells |
|
| - Suspension Cells No-Spin Procedure |
Add 200 ml/well Fixing Solution on top of the cells. Incubate 1 hour at Room Temp Aspirate the Fixing Solution and blot the plates dry. |
| - Suspension Cells Spine Procedure |
Spin the plates for 5 minutes at 1000 rpm. Aspirate media, add 200 ml/well Fixing Solution. Incubate for 30 minutes, room temp. Aspirate the Fixing Solution and blot the plates dry. |
| 7. Wash Step | Wash X3 with 1X wash buffer and blot dry. |
| 8. Detector Antibody | Add 100 ml/well of diluted detector antibody. |
| 9. Incubate | 1 hour at room temp. |
| 10. Wash Step | Wash X3 with 1X wash buffer and blot dry. |
| 11. Conjugate Addition | Add 100 ml/well HRP-conjugate |
| 12. Incubate | Incubate for 30 minutes at room temperature. |
| 13. Wash Step and Final Water Wash | Wash as above. Perform a final distilled water wash by flooding the entire plate with distilled water. Pat dry on absorbent paper towels. |
| 14. Development |
Add 100 ml/well TMB Peroxidase substrate |
| 15. Incubate | 30 minutes at room temperature in the dark. |
| 16. Stop | Add 100 ml of acid Stop Solution to every well |
| 17. Read |
Read
the at 450/550 nm
|
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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics