Viability of sorted cells

Eckstein, Volker Volker_Eckstein at
Tue Jul 31 07:05:52 EST 2001

Dear flowers and sort specialists,

I have some problems sorting viable CD20 pos. cells from multiple myeloma
patients blood.
Technical details:
Cells were isolated by Ficoll Hypaque gradient centrifugation and labelled
with anti CD20-FITC monAb and kept in ExVivo medium with 20% FCS on ice
prior to sorting. Percentage of CD20 cells in sample to be sorted: 10-20%
Viability of the samples were about 96% as shown by trypan blue and PI dye
Sorting was done on a FACS Vantage SE with turbo sort.
Settings: Sheath pressure 20psi, ddF 46400hz, sample diff. max. 0.6psi.,
flow rate 5000-6000 events, nozzle 70micron, sorted drops 1.2, abort
Cooled sorting devices: normal facs-tubes with 3ml medium to ensure to catch
the sorted cells and keep them in best condition. No sterility problems in
the following cell culture.
Reanalysis of the sorted cells showed the desaster. Purity of sorted cells
(CD20 pos. cells): 96% (fine).  But between 50% and 65% of the sorted CD20
cells shifted beyond the FSC-threshold of viable cells and were PI positive.
The expression of these CD20 cells was lowered after the sort by at least
half a log or up to nearly zero. Different cells sources (patients) showed
different extent of antigen loss in this area - some more, some less. Viable
cells fitted to the original sort gates and were not altered in fluorescence
intensity and scatters.
What is the sorter doing with these cells? Any suggestions? Thanks in
advance  from a frustrated operator.

Best Regards	Volker

Volker Eckstein PhD
Dept. of Internal Medicine
University of Heidelberg
Hospitalstr. 3
69115 Heidelberg
Phone:	 +49 6221 567356
Fax:		+49 6221 565775
volker_eckstein at

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