[Cytometry] storage of fixed cells after brdu incorporation

Jaromir Mikes jaromirmikes at yahoo.com
Wed Apr 10 14:25:38 EDT 2013


Hi Keith,

When you use 70% EtOH and you close your tubes well, or even use the parafilm to avoid evaporation, you can store them for longer period. The "overnight" storage at 4°C or -20°C is the "minimum" to make sure that the cells will be fixed well (3-4 hours/RT are enough for most cells). You can keep your samples for weeks, similarly to DNA/PI staining for cell cycle, but its always better to handle them gently and don't let them wait too long. Avoid mechanical stress, don't pipette them vigorously, use rather milder and longer centrifugation steps and your sample should love that. >;o)) They will have, though, some hard times during the harsh DNA denaturation step.
 
Good luck

Jaromir

RNDr. Jaromir Mikes, PhD.
Institute of Biology and Ecology
Faculty of Science
P.J. Safarik University in Kosice
Moyzesova 11
040 01 Kosice
Slovakia
tel.: +421(0)55-234-1205


________________________________
 From: "KDT7 at pitt.edu" <KDT7 at pitt.edu>
To: cytometry at lists.purdue.edu 
Sent: Wednesday, April 10, 2013 3:14 PM
Subject: [Cytometry] storage of fixed cells after brdu incorporation
 
Hi all,
I am performing brdu pulse-labeling for S-phase analysis. After
incorporation of brdu and subsequent fixation (I am using 70% ethanol), I
am wondering if it is possible to store the cells (say, at 4 or -20°C) for
later analysis, or if its best to continue immediately with the rest of
the staining protocol? Many of the protocols I have seen do not mention
storage (the longest I have seen is overnight at 4°C). When doing PI DNA
stains, I have had good luck storing the cells in ethanol at -20 prior to
the PI stain,  although brdu is a different ballgame. Thanks in advanced
for your help!

Best Regards,
Keith Task
University of Pittsburgh

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