EDTA kills 293 cells. simply pipet them out from the plate, with filtration through a nylon mesh if necessary. 10% serum in the staining medium extends viability. DNase would reduce clumping due to dead cells. Saverio Alberti Head, Lab. of Experimental Oncology Department of Cell Biology and Oncology Consorzio Mario Negri Sud 66030 Santa Maria Imbaro (Chieti), Italy Phone: (39-0872) 570.293 FAX: (39-0872) 570.412 E-mail: alberti@cmns.mnegri.it On Mon, 6 Mar 2000, [iso-8859-2] Boros András dr. wrote: > > Dear colleagues, > > I struggle with stable transfected HEK-293 cells. I need to compare the > expression level of a certain (transfected) receptor on these cells. I have > difficulties with the extended aggregation that take place during the > labeling procedure which affects mean fluorescence. Do you have any good > protocol to decrease the extent of aggregation, or at least standardize it. > > Thanks for your help, > > Best whishes, > > Andras Boros > > > My protocol is : > Suspension of cells with EDTA containing D'PBS, wash and fixation for 20 min > in 4% PF. > Then labeling with primary antibody O/N, wash and labeling with > FITC-conjugated secondary antibody (2.5 h), wash and analysis with FACScan. > (LOG amplification of all parametrs) >
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