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cells from dissociated tecta. Purified cells were routinely
>99.99% A2B5(-) as assayed by indirect immunofluorescence.
Also, the vast majority of cells recovered from the
microspheres were low level A2B5(+). Yields of A2B5(-)
cells have ranged from 100% of theoretical yield (50% of
total cells) to less than 50% of theoretical yield,
depending on the batch of microspheres used. M450.40
microspheres gave the highest purified cell yields, while
M450.51 and Dynabeads M450 gave significantly lower yields,
presumably due to increased nonspecific binding to or
trapping of A2B5(-) cells.
For separation of cells via A2B5, I have found it
necessary to use a polyclonal antibody that was
specifically produced for the purpose of microsphere
coating. Several commercial affinity-purified polyclonal
anti-mouse antibodies have been tested for effectiveness
with this monoclonal without satisfactory purifications.
Microspheres precoated with anti-mouse IgG (Dynal) have
also been tested with similar unsuccessful results. The
results presented here represent only experiments where the
purity of A2B5(-) cells exceeded 99%.
A2B5 Antigen Modulation
Although the separations resulted in purified A2B5(-)
cells, it was found that many A2B5(+) cells appeared after
in vitro culture for only several hours. After one day in
culture, approximately 50% of the initially purified cells