As far as I know there are no cons. We have done this for intracellular staining for 6-7 years on a bizillion samples without a problem. There does not appear to be any added noise due to the freeze-thaw. After fixation the cells are diluted in a 4 fold excess of PBS with 0.1% BSA and then centrifuged at 2600 rpm (approx. 750 x g?) on a table top Sorvall cell culture centrifuge. The pros are that you can freeze down replicates and : 1. be able to disassociate the stimulation from the staining experiment; 2. can batch samples stimulated on different days; 3. have room for error; 4. be able to look at additional cytokines and markers after your first experiment or even years later. Additionally, we perform all of our single color controls using unstimulated fixed PBMC. We fix a billion or so PBMC and then aliquot 10 million to a tube. Comes in very handy for compensation as well as for titration experiments for cell surface markers. We use PBS with 10% DMSO. Don't need to use fancy cell culture grade DMSO, as the cells are dead! Surfs up, gotta' go Calman > ---------- > From: Heather Medbury > Sent: Wednesday, May 30, 2001 20:05 > To: Cytometry Mailing List > Subject: How do you freeze cells for storage prior to staining? > > > Hi FLOWers, > > > We are planning on doing intracellular cytokine staining, I saw in the BD > application > note on 'Decting intracellular cytokines in activated lymphocytes' a brief > comment > about being able to store stimulated samples at - 80°C and then stain at a > later time. > > Though we do plan on staining our samples straight away, it would be nice > to store > the excess cells away. > > Do any fellow FLOWers freeze their stimulated samples away for later > analysis. If so is > it for intracellular staining or what other applications can this method > be applied > to, and what is your freezing and subsequent staining protocol? what are > the Pros / > cons etc. > > > > Thankyou in advance, > > > Heather > > > Heather Medbury (PhD) > Senior Scientist > Department of Surgery > Westmead Hospital > Westmead 2145 > 61-2 9845 7677 > -------------------------------------------------------------- > All experiments are preliminary > > ------------------------o0)(0o-------------------------------------- > > "In his heart a man plans his course > but the Lord determines his steps" > > --------------------------------------------------------------- > > >
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