Saving tissue and single cell suspension - FACS, single cell suspension (May/26/2010 )
I have some questions about the basics of FACS
well, I want to prepare the single cell suspension of lung tissue, but as the conditions in my lab, is it ok to keep the lung tissue in FCS/EDTA/PBS (or just PBS) in ice for about 1hr before preparing the cell suspension?
Will the cell dead easily under this conditions?
and, if I do the secondary staining to the 1st antibody, is it better to mix the 1st Ab and 2nd Ab at one time, or stain respectively after wash and centrifugation?
Many thanks to your reading and answers
1st time 1st, if u doing FACS sorting to isolate cell for downstream experiment or cultivation, do it fast
otherwise, it shud be ok to keep for an hour or two(sure will have dead cell, coz non-media buffer)
the answer is no
for the sake of easiness, get a primary with flourecent
cheers and happy flowing