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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