Fellow users:
Your experience would be greatly appreciated in helping us overcome cell
clumping during our sorts.
We start with frozen mobilized blood stem cells and thaw in the presence
of DNase in Ca/Mg-free medium. The CD34+ cells are enriched with magnetic
beads. The resultant CD34+ fraction is left overnight in tissue culture
medium with cytokines, during which time cell clumps arising from latent
post-thaw damage disaggregate. However, when the cells are stained the
following morning, cell clumping occurs again, aggravated no doubt by the
pelleting and washing steps. We keep the "vortexer" running continuously
during the sort (Coulter Elite), but it does not noticeably prevent the
formation of large aggregates. We are using a 100 micron flow cell now. In
spite of these precautions, the formation of clumps is playing havoc with our
sorts!
Does anyone have a suggestion? Has anyone ever used basic additives
(spermine, poly L-lysine, or just L-lysine) to overcome clumping? Would such
an approach increase the loss of cells to glass and plastic surfaces? We
have heard that cell clumping is reduced at pH's around 5.0 -- would this
interfere with (1) cell viability (2) antibody-antigen binding (3) change
fluorochrome excitation/emission characteristics or (4) decrease
antibody-fluorochrome stability? Your thoughts and suggestions would be
appreciated. Thanks!!
Doug Dooley
Stem Cell Laboratory
American Red Cross, Portland, OR
phone: 503-280-1442
E-mail: DEEKEWB@AOL.COM
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