We are trying to measure the proliferative capacity of adherent monolayer adult rat hippocampal neural stem cells under differentiation conditions. We plated the cells at recommended density (25,000/cm2) on PLO-LN coated plates, and induced differentiation by withdrawing bFGF from culture medium. However we observed a higher rate of proliferation (measured by SIGMA Cell Proliferation ELISA-BrdU) on induced conditions (no bFGF), compared to non-induced conditions (20ng/mL bFGF). Could this be because the labeling time was too long (12 hours)? We also incubated the cells for 24h before labeling, or is the incubation duration too long/too short?
i hope you are aware that BRDU staining requires treatment with an acidic solution to enable antibody to bind. here is our protocol for use with freely floating sections. use of another antibody or different fixative will require careful titration
Thank you very much for you answer. We're using SIGMA Cell Proliferation ELISA, BrdU kit which has a ready-to-use FixDenat solution which is acidic.
For differentiation you need to remove both EGF and FGF. We fix mouse NSCs after 30minute incubation with BrdU. You may need to test different times. With a 12-hr incubation you may be picking up 'unhealthy' cells or cells going thru DNS repair by your labeling strategy.
I used 90 minutes for brain brdu staining following ip brdu injection in mice. I guess 30'-60' is the optimal range for in vitro studies
BrdU incubation time depends on how rapidly the cells divide. Primary cells may need up to 24 hours, while rapidly proliferating cell lines may only need one hour. The exact time required to achieve the optimal signal-to-noise ratio should be optimized.
Good luck.
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