I've actually had a smiliar thread before with no direct answers regarding the fluorescence dragup :( I wonder if its the protein content in the solution? Or number of washes after the lysis? I was toying around with trying a plethora of configurations on the buffer but I thought I would consult the group for some working buffers, specifically with regards to the change in signal strength issue. Maciej ----- Original Message ----- From: "bcotleur" <bunny@cotleur.com> To: cyto-inbox Cc: "cytometry" <cytometry@flowcyt.cyto.purdue.edu> Sent: Tuesday, June 25, 2002 7:58 PM 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!***** >
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