We have recently started trying to use BrdU/PI
flow to study the cell cycle characteristics of embryonic mouse brains. We have had
some success in obtaining single cell suspensions but are having real problems with
cell loss during BrdU staining which we think is related to HCl treatment to denature
the DNA (could this be lysing these fragile cells?). 70% ethanol fixed cells treated
with 2N HCl for 30mins show good BrdU staining under the microscope but are really
very few (90%+ lost)
Has anyone done such an analysis?
Does anyone have a suitable protocol?
Or know anyone who does?
Or any good ideas?
We look forward to any helpful suggestions.
Paulette Zaki and Ben Martynoga
Department of Biomedical Sciences
University of Edinburgh