 |
 |
 |
 |
I do not know what kind of cells you are trying to stain, but there are two main
areas that can give problems in this procedure:1) denaturation of DNA, and 2)
fixation and permeabilisation of your cells. If HCl denaturation given in the BD
protocol is not working, you may want to try heat denaturation or increasing the
time of HCl treatment. I am working with PBMC and use DNAse for denaturation
(because I need to maintain cellular protein structure). I use FACSlysing soln.
for fixation followed by FACS permeabilisation soln., both made by BD, and it
seems to work well. You may need a higher conc of BrDU (eg. 50 mcM instead
of 10) or a longer incorporation time depending on what cells you are working
with. Also, you might want to monitor your cell population for some loss of
cells (loss of propidium iodide staining) which can be seen when sufficient
denaturation occurs. Hope you find the info. helpful.
Bela Mehta
Post-doctoral Scientist
Becton Dickinson Immunocytometry Systems
San Jose, CA
|
|
|
|