cell cycle analysis by BrDu FACS

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ameybendre
ameybendre's picture
cell cycle analysis by BrDu FACS

Hi,
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.
bye.

samm
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.