Bunny, We routinely fix samples in 4% PFA and then store in PBS/10% DMSO at -80 C. The cells are good for OVER A YEAR and I would think almost forever. We use these in our intracellular staining for allergen specific T cell responses. I have not see any evidence of major increases in autofluorescence or non-specific binding. I think these allergen specific responses are some of the less frequent intracellular events that have been detected (0.01-0.1% of CD4 T cells). Our "background" in the isotype stained samples or in cytokine stained media control samples is typically 0.002%...even after a year. Are your cells that are gaining background frozen at -80 or in the frig? We make up PFA in 2-3 liter batches and the freeze in 15 ml aliquots (-20 C) that are kept at 4 C and then tossed 1 week after thawing. I'm happy to send you our recipes. Calman > ---------- > From: bcotleur > Reply To: bunny@cotleur.com > Sent: Tuesday, June 25, 2002 22:58 > To: Cytometry Mailing List > Subject: Re: help - fixative issue? > > > Macie et al- > This brings up another hot topic-- the length of time fixed samples can be > successfully analyzed. Richard Meister just posted that he can store > fixed > samples for ONE YEAR. (My hat is off to you Richard- but I have never > been > that lucky). Loss of fluorescence wasn't my problem- it was the increased > auto/background fluorescence that haunts me. > > Over & over I've seen in papers/emails /posted protocols that fixed > samples > can easily be stored for periods of time ranging from days to weeks (and > now a > year). Yet I have witnessed exactly what you posted in your attachment. > Within > 24 hr I can pick up subtle increases in fluoresence. By 3 days I usually > have > to decrease the PMT's so much/increase the comp tons/ I doubt the > validity of > some of my positives-especially the dim/"shoulder" type positives. (I'm > generally running human lysed whole blood or PBMC's, and just for the > extra > 2cents- the scatter actually looks great). I also notice that the > monocytes > seem to brighten/move up the scale much more than the lymphs, and that on > the > unstained/control samples FL1 shifts the most, the Fl3, then Fl2. > > I've tried every protocol I've seen: 0.5%,1%,2% fresh PFA, with and w/o > post > fix washing in PBS/2%serum; 0.5%, 1%,2% EM grade formaldehyde (both > stored in > the fix and also washed after fixation just like the PFA). > I've scrupulously monitored the pH, kept the stuff in brown bottles, made > small batched and big batches,used it fresh and used it 1 week old- I > don't get > the voodoo involved here. > I now stick with the EMgrade formaldehyde from Polysci(1%) and run my > samples > <36hr. Since I'm running chemokine analysis-(which often are not > completely > separated positives such as Tcell markers) on patients on Rx Tx-I'm not > comfortable having the cells shifting up during storage and figuring out > what > is artifact and what is response to Tx. > > If anyone out in flow-land has the secret key to fixation- I would love to > hear > it. And if there is a trick to analyzing samples with increased > background > (due to storage)-I would love to be enlightened! > > --bunny--- > ******all fixed up & nowhere to go!***** > > > > maciej simm wrote: > > > Dear group, > > > > In the attached word file (virus free to the best of my knowledge) I am > > showing two acquisitions of the same file. It was fixed in the 0.75% PFA > > with 1% HAB serum final in PBS recipe. I don't think its working because > the > > same instrument settings show a huge difference in fluorescent > distribution. > > Does anyone else experience this drag-up with their fixatives? are there > any > > fixatives which don't do this as much? > > > > > ------------------------------------------------------------------------ > > Part 1.2Type: Macintosh File > > -- > Bunny Cotleur, M.S. > Sr. Technologist > Cleveland Clinic Foundation > Neurosciences NC30 > 9500 Euclid Avenue > Cleveland, OH 44195 > cotleur@ccf.org > (216) 444-1164 > fax (216) 444-7197 > > > >
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