cell cycle analysis by BrDu FACS

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ameybendre's picture
cell cycle analysis by BrDu FACS

i want to do cell cycle analysis by using BrDu FACS.but i have apo-BrDu kit of BD.can i do my experiment with that kit? or i have to follow different protocols ? pls also tell me proper protocol of BrDu cell cycle analysis.
thanx in advance.

samm's picture
You don't have to use the

You don't have to use the expensive $7/sample apo-Brdu for proliferation. Pulse your cells with 0.1 mg/ml BrdU, pH7.4 (Sigma) for the last ~15 h (variable depending on cell type -range 8-20 h) of your selected expt duration. If you have no idea about how long to track your cells under a specific treatment condition, just do 24, 48 and 72 h to start, with a 15 h pulse included.
Harvest cells, fix with 4% paraformaldehyde (pH7.4) or 10% neutral buffered formalin according to the intrac FACS staining protocols on this site. Do the anti-BrdU-FITC/APC or eq staining using the same protocols, wash well, add the PI in 0.11% citrate buffer+RNAse, stain o/n and acquire. Note that the intense PI staining will be detected on both FL2 and Fl3 channels of BD cytometers - try to keep both clear.
The BrdU+ popln gives you cumulative DNA replication, PI gives a snapshot of hypodiploid(dying), G0/G1 (stasis) or S/G2M (actively dividing cells) at that time point.
For a better cumulative analysis, consider using CFSE, DII or equivalent dyes instead of BrdU.