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Kevin G. Waddick, Ph.D.
University of Minnesota
Dept. of Therapeutic Radiology and Radiation Oncology
Biotherapy Program
Box 494 UMHC
Minneapolis, MN 55455
(612) 625-1180 Office
(612) 625-7060 Fax
On Fri, 30 Jun 1995, Penney Koeppen wrote:
> Has anyone used FACS analysis of liquid cultures of CD34+ cells from cord blood
> to reproduce data obtained from parallel methylcellulose colony assays? eg:
> Would the number of BFU-E's in methylcellulose correlate with % glycophorin A-
> positive cells in a parallel liquid culture; and would the number of CFU-meg's
> correlate with % CD41a-positive cells?
>
>
One should keep in mind that the immunophenotyping results obtained
from bulk culture include the expression of end-stage lineage-restricted
antigens that are not present on the more immature clonogenic precursor
cells. While there may be large numbers of mature cells sloshing around
unattached in liquid culture (this being totally dependent on the
combination of cytokines provided), the progenitor cell that gives rise
to a particular type of colony is immature and expresses the CD34
antigen; the expression of CD34 and end-stage antigens are often mutually
exclusive.
How long after harvest and the length of ex vivo culture in
the presence of defined cytokines would play a role in the similarity of
the phenotypic results with the colony counts and lineages derived from
colony assays; the longer the period, the more non-mitotic end-point
cells would apoptose, thereby causing the quiescent Go clonogenic
precursors to represent a higher proportion of the remaining
population.
Of possible interest is my experience with immunophenotyping cells
removed from methylcellulose colony assays. It is not possible to do
this and get meaningful percentage results. The problem is that the
cells are very sticky after being in methylcellulose and they bind
antibodies nonspecifically. In other words, the level of staining with
MsIgG1-FITC is so high that setting a quadrant based on this to determine
CD34-FITC positivity might give artifactually low values, because even if
a large fraction of the cells display CD34 it would only boost the
intensity of staining marginally and with no separation from the
background control.
Kevin G. Waddick, Ph.D.
University of Minnesota
Dept. of Therapeutic Radiology and Radiation Oncology
Biotherapy Program
Box 494 UMHC
Minneapolis, MN 55455
(612) 625-1180 Office
(612) 625-7060 Fax
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