10
inactivated FBS (HI-FBS) for 30 min. at 4C. Cells were
then rinsed 2x in Tyrode's and resuspended in SBTI-DNase.
The A2B5 hybridoma cell line was obtained from the
American Type Culture Collection, Rockville, MD, through
Dr. Michael F. Marusich, Univ. of Oregon, Eugene, OR.
Cells were mixed for 30 min. at 4C in SBTI-DNAse + 10% HI-
FBS with paramagnetic polystyrene microspheres (4.5 (J.m;
Dynal, Inc., Great Neck, NY) which were previously coated
with sheep anti-mouse IgG prepared as above. A 15-fold
excess of the number of microspheres/ the number of A2B5(+)
target cells was used which corresponds to a 7.5-fold
excess of microspheres/ total cells, since approximately
50% of dissociated cells were A2B5(+). The microspheres
were ethanol sterilized then coated with 30-40 ug antibody/
mg microspheres in a concentration of at least 0.2 mg/ml
antibody overnight at 4C with rotation in a microfuge
tube.
The cell-microsphere mix was then poured into the top
of the separation chamber which was prefilled with
Tyrode's, and unbound cells were eluted with Tyrode's by
gravity at a rate of approximately 1-2 ml/ min. in a
sterile hood until no more cells appeared in the eluant. In
a typical experiment, approximately 3xl07 cells were
separated in 30 min. The separation chamber was constructed
from a plastic funnel and tubing (1/4" dia.) held on a ring
stand (Fig.2-1). Two magnet arrays were held against the