John, ? It is a natural phenomenon that PHA stimulation of PBMC results in cluster formation, as phytohemagglutinin agglutinates blood cells (according to its name). Moreover, polyclonal cell activation results in massive upregulation of adhesion receptors, thus these cells stay together after division and will also exercise increased stickiness during sample processing. Nevertheless, we have routinely used mitogen stimulated cells in flow analysis, so as many others, especially since inception of division tracking dyes. Some points for handling stimulated cells: ? i) Careful pipetting while harvesting cells from culture plates is usually enough for disrupting the clusters. The effect can be controlled by microscopy or simply observing cells in wells in an inverted microscope. At the minimum, one should check the disrupting effect while establishing one's routine resuspending technique. In case of clinical diagnostic procedure, we specified the number of times for expelling the content of a well and pipette volume to standardize resuspension in the SOP. ? ii) Make sure the sample is resuspended before acquisition on a flow cytometer, as the cells may also clump during staining and washing/centrifugation. This can also be checked in a microscope, especially if the flow picture casts any doubts. ? iii) Doublet discrimination strategies have to be in place, so as exclusion of occasional "too large" events. ? Merry Holidays and Happy New Year to every one! ? Vladislav Rozenkov -----Original Message----- From: Michie, John, Dr <jm5@sun.ac.za> <jm5@sun.ac.za> To: cyto-inbox Sent: Thu, 13 Dec 2007 8:05 pm Subject: PHA stimulation of PBMCs results in sticky cells 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 ? ________________________________________________________________________ More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com -- End --Received on Mon Dec 17 12:38:00 2007
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