BrdU Proliferation (Colorimetric)

	Frontier™ Bromodeoxyuridine (BrdU) Cell Proliferation ELISA Kit
Exalpha's Frontier™ 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)
	Ordering Information
	Cat. No. X1327K1 X1327K2 X1327K3	Size 200 Tests 1000 Tests 5000 Tests
	For larger quantity pricing, please contact Customer Service

Legend:
Exalpha’s Frontier™ 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.

Protocol Summary: Not to be used in place of detailed protocol. See kit manual for complete protocol.

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