Adult stem cells isolation from bone marrow

Ayelet Ben David ayeletben at ekmd.huji.ac.il
Thu Jan 25 04:37:46 EST 2007


Hello good people,


We are trying to Immune Isolate human mesenchymal stem cells (hMSCs) from
bone marrow by CD105 marker.
Therefore we are using the Auto MACS with miltenyi CD105 micro beads then
CD105 PE conjugated antibodies for FACS analysis from Ancell.
As a control for the nonspecific binding, we use IgG1 PE Isotype by Ancell.
We expect to see ~2% of CD105 in the mono nuclear cells (MNCs) group, as we
see. 
We also expect to see above 50% of CD105 in the CD105 positive group (After
Isolation by MACS), but we see only ~5%.
My question is why we see this low signal situation after the MACS
isolation.

We suspected the antibodies to be expired, therefore has low affinity but
it's not the problem.
We suspected the Micro beads to block the binding site but were told by
miltenyi support that it uses only 30% of the epitope binding site.

For clarification, our lab had positive experience in the past with MACS
isolations and FACS analysis.
I would appreciate it if you could help me with that.

Ayelet Ben Arav 
M.Sc. student
The Hebrew University of Jerusalem
Email: ayeletben at ekmd.huji.ac.il






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