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 exclusion. 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 rate:15-20% 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 GERMANY Phone: +49 6221 567356 Fax: +49 6221 565775 volker_eckstein@med.uni-heidelberg.de ********************************************************************** This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please notify the system manager. This footnote also confirms that this email message has been swept by MIMEsweeper for the presence of computer viruses. www.mimesweeper.com **********************************************************************
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