From: Dave Coder (dave@nucleus.immunol.washington.edu)
Date: Mon Jan 31 1994 - 12:18:10 EST
Begin forwarded message: X-Nupop-Charset: English Date: Mon, 31 Jan 1994 08:55:38 +0100 (CET) From: Dr Stephen Young <s.p.young@bham.ac.uk> Sender: steve@rheuma.bham.ac.uk Reply-To: s.p.young@bham.ac.uk Return-Receipt-To: s.p.young@bham.ac.uk To: KWEBER%IMVS4.decnet@uv1.im.med.umich.edu Cc: cytometry@flowcyt.cyto.purdue.edu Subject: RE: BIG FILES ... We are also measuring calcium fluxes but have kept away from attempting simultaneous surface labelling becuase of the possible effects of the antibody on the subsequent calcium flux. How do you get over this? and which surface proteins are you labelling? ... For the last several years we've been measuring calcium flux along with two surface markers on cells from mouse thymus, spleen, or peripheral blood. Antibodies against CD4 and CD8 are routine, and we've done some with three surface markers (FITC, PE, and PE-Cy5 conjugates all excite with 488nm light). Calcium flux has been induced with agents including anti-CD3, phorbol esters, or peptides. Surface-labeled cells when probed with ionomycin display an indo-1 violet/blue ratio that is 12-16 times greater than the resting state. Data files become large, necessarily so since some phenotypes (which you can identify directly with multiple surface markers) are present at low frequency. We can read and analyze these multi-megabyte files quite easily with ReproMan. Dave Coder Dept. of Immunology Univ. of Washington dcoder@u.washington.edu
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