Our Flow Cytometry Core facility at MD Anderson Cancer center over the year had about a dozen users who were interested in measuring the expression of Green fluoresrescence protein in conjunction with another phenotype either a surface marker, a marker for cell cycle distribution or a marker for apotosis. In most experiments, the cells were transfected with GFP, used as monitor of transfection, and a gene of interest were hematological cell lines or epithelial cell lines (HL60,MCF7, ovarian cell lines). The idea was to study if the expression of the gene of interest changed the expression of the surface marker, cell proliferation or apoptosis. GFP being used to detect/prove successful transfection of the gene of interest, briefly a function assay for the latter. 1)We have found that in all cases GFP were destroyed by even very mild detergent(Tween 20 at 0.1%), Alcohol(Ethanol, Methanol) 2)Paraformaldehyde at 0.5% to 2 % preserved the expression of GFP at least when comparing the percent of cells expressed before and after addition of PFA. The separation is about half a log poorer when Median /mean channel number of the transfected and untransfected population before and after addition of PFA was compared. When the initial separation was 2 logs, this was not a big problem, but if the GFP positive population was only a smear barely above the background then the functional assay provided ambiguous results. 3)When DNA profiles were required, we found that Propidium iodide gave very low resolution profiles, i.e., CV of 6% or more but Hoeschst33242 worked if you have dual laser system equipped with a well aligned UV laser. 4)Apoptosis assay using Tunel with fitc labeled nucleotides and exogenous terminal transferase was slightly affected. The DNA profile with Hoeschst 33342 were good enough to quantitate apoptotic cells when apoptosis was well advanced not in early apoptosis. We are trying Anexin both green and red Alexal with , still, disappointing results. Hope that these will help Ntvan(Not yet a guru!) Fluorescent protein aaa001@agora.ulaval.ca wrote: Dear Flowcyte gurus, First I would like to thank all for their good advice with paraformaldehyde. I found this List a week ago and really appreciate the quality of the discussion and information. I already searched the archive for my other question, but found nothing about it. So here is my question : I am studying the expression of constitutive GFP in mammalian cells. GFP will leak only from dead cells when membrane integrity is loss (personal observation). As paraformaldehyde permeabilize the cell membrane, will the GFP expressed will be entrapped into cells or will GFP leak with time... Is there any way to prevent the leakage of GFP after fixation ? Does high paraformaldehyde concentration could be the solution (4%, 6%, 8% ?). Thank you for your help. Philippe-Alexandre ™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Philippe-Alexandre Gilbert Institut de recherche en biotechnologie Conseil National de Recherche du Canada 6100 Royalmount, Montreal, Quebec (Canada) H4P 2R2 ____________________________________________________________________ More than just email--Get your FREE Netscape WebMail account today at http://home.netscape.com/netcenter/mail
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