BrdU Proliferation

BrdU Proliferation Assay is a completely HTS compatible, non-isotopic, colorimetric proliferation enzyme-linked immunosorbent assay (ELISA) assay kit for the detection of bromodeoxyuridine incorporation into newly synthesized DNA of adherent and non-adherent cells.

Features:

- Colorimetric assay
- HTS compatible format
- Optional spin step
- High sensitivity
- 4 deg C storage with long shelf life
- Non-radioactive
- 2.5 hour protocol
- Suitable for all species
- Suitable adherent or non-adherent cell types

Performance Summary:

- Sensitivity: 40 cells/well
- Intra-assay C.V.'s: <10% (spin protocol)
- Inter-assay C.V.'s: <10% (spin protocol)

Legend:
BrdU assay. Detection of variable numbers of Jurkat (non-adherent) or CHO cells (adherent) per well. Y axis-left, OD 450-550nm. Y axis-right, signal-to-noise ratio.

1. Cell Plating � no Test Reagent/Drug (skip step 3 below)	� 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	� Aspirate (or flick) the media from the cell wells � Add 200 ml/well Fixing Solution � Incubate 30 minutes at Room Temp. � Aspirate the Fixing Solution and blot the plates dry.
- 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