I received the following message which I thought I would pass on to the community.... Please reply directly to the address below with answers... thanks Paul ------------------------------------------------------------------- From: Yasser M El Sherbing <yelsh001@umabnet.ab.umd.edu> To:robinson@flowcyt.cyto.purdue.edu Hi I got your e-mail from the enternit and i have a problrm with the flowcytometry i am trying a new drug on human breast cancer cell line 231 I incubate the cells both the control and the drug treated cells with 10um bromodeoxy uridine for half an hour followed by washing, trypsinization of the cell washing twice then fixation in 70% ethanol at 2 degree centigrate then on the day of running them in the flowcytometry usually within 2 or 3 days i denaturate the cells with 3 n.Hcl then i add Na tetraborate then wash twice with pbs/albumin/tween 20 then i adjust the cell number to be 1 million/1ml pbs then i add 20uL of anti Br.deoxy uridin antibody fitc labelled and incubate for half an hour then wash twice and then adjust the cell number to be 1 million / ml pbs then i add propidium iodide 100 uL/ 1 million cell . the strange thing that the Pi result is very good i get a good histogram with G1, S and G2m but for the BR.d.u nothing appear both the control [mouse IgG] and the Fitc labelled antibody is the same very dim nothing specific as if there is no BR.d.uridine preasent i am using coulter flowcyt.. wave length for the bromodeoxy uridine is 525 . the anti-br.d.ur. antibody Fitc from becton dickinson, the bromodeoxy uridine from boeheringer. i tried different conc of br.d.ur 20 & 30 ul i always obtain the same results I tried to use a highier conc of Hcl but it killed the cell and i got a lot of debris so i hope i could find some help my e.mail yelsh001@umabnet.ab.umd.edu -------------------------------------------------- this message was bounced to cytometry by J.Paul Robinson, Purdue University Cytometry Labs Professor of Immunopharmacology robinson@flowcyt.cyto.purdue.edu PH:765-494 6449 FAX:765-494 0517 web http://www.cyto.purdue.edu
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