purification of CD34 cells

From: andreas.simm@medizin.uni-halle.de
Date: Fri Dec 06 2002 - 07:46:44 EST

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Hi experts,
we have problems to isolate CD34 cells.

We started with sodium-heparin blood,
mixed with MACS buffer (EDTA, BSA, PBS)
and isolate PBMCs with a FICOLL gradient.

Thereafter we used the AutoMACS to
purify CD34 cells. Unfortenately, we only
get purities about 70%. We tried a lot
(direct CD34 isolation, indirect method,
depletion of CD14 / CD15 cells) but
we never get a reproducible high
purity. In addition, the guys from
Milteny tried to help us as well, but
until now, we could not find the fault.

Who has experience with this method
and can help us. Should we try to add
an additional purification of lymphocytes
(and the CD34 cells within) before starting
the MACS? Should we include a DNAse step
against clumping (but we do not see them)?

Thanks for any advice

Andreas






PD Dr. Andreas Simm
Universitaet Halle Wittenberg
Klinik fuer Herz- und Thoraxchirurgie
Ernst-Grube Str. 40
D-06120 Halle

Tel.: +49 (0) 345 557 2647
                  552 2878
FAX:  +49 (0) 345 557 2782
                  552 2890

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