Sorting of human WBC after freezing

From: Brendel Cornelia (brendel@oncocenter.de)
Date: Fri Oct 17 1997 - 08:59:23 EST


Hi,

I am working with a FACS Vantage with TS option in a hematology/oncology
lab. We are sorting stem cell subpopulations in human leukapharesis
products. Some of the cells are frozen in plasma and 10 % DMSO like
normal blood cells for stem cell transplantation.  After thawing in warm
water I quickly dilute the cells in PBS with 5mM EDTA and 10 %FCS and
wash them. But within a short time (about half an hour) cells will
agregate so that staining and sorting is impossible. Has anyone
suggestions how to avoid this clumping? I have tried trypsin and nylon
mesh to disconnect the cell masses, but that was not sucessful. Any
proposal is highly welcome since we have deepfreezed many interesting
samples from leukemia patients.

Thanks in advance,

Conny Brendel



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