On Thu, December 13, 2007 3:05 am, Michie, John, Dr <jm5@sun.ac.za> wrote: > A colleague has the following problem: > > After stimulation of PBMCs with PHA the cells become sticky. Trying to > analyse on flow shows reduced numbers of cells compared to fresh. > > Cause of stickiness? > > Any help flowers? > > > > Kind regards > > John Michie > > +27 21 9389 539 > > +27 21 933 8886 > > 083 449 2225 > > > > PHA (phytohemagglutinin A) causes aggregation of red cells and also white blood cells to a considerable extent. PBMC is purified over Ficoll to primarily enrich mononuclear cells. We routinely culture PBMC with PHA and notice that a fraction (5 to 10%) will die by apoptosis within a day or two. However, if you want to analyze the cells by flow cytometry, it is easy to disrupt the clumps using a glass or disposable pasteur pipet. Pipet 3 or 4 times, collect the cells, centrifuge and resuspend in ca, Mg-free Dulbecco's PBS (Invitrogen) + 0.2% BSA to minimize clumping. Cells can be stained in this buffer (+ 0.01% sodium azide) on ice for 20 minutes, washed with the same buffer, fixed in 1% freshly prepared paraformaldehyde and analyzed without any problem. SJ -- S. Jayaraman, Ph.D. Associate Professor of Surgery University of Illinois at Chicago 909 South Wolcott Avenue-704E MSB-M/C 790 Chicago, IL 60612 Phone: 312-355-5133 Fax: 312-355-1497Received on Fri Dec 14 12:23:56 2007
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