>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
You might try using some DNAse in your media as the clumping may be caused
by DNA from cells that have been destroyed through manipulations.
Roger A. Burger E-mail: Roger@cpd2.usu.edu
Research Associate, Immunology
Center for Persons with Disabilities
Utah State University
Logan, UT 84322-6895
Voice: 801-797-2042
FAX: 801-797-4054