11
side of the tubing, one made of ferrite magnets above one
made of samarium-cobalt magnets. A pinch clamp at the
bottom of the tubing was used to control the rate of flow
induced by gravity.
Bound microsphere fraction cells were collected by
first removing the magnet arrays from the side of the
tubing and then washing out the bound cells and
microspheres into a test tube. Cells were released from the
microspheres by trypsinization as above followed by
addition of SBTI-DNAse and vortexing. Freed microspheres
were drawn away from the cells by placing the samarium-
cobalt magnet array against the side of the tube and the
suspended cells were aspirated out of the tube with a
pipette.
Cells to be assessed for cell surface A2B5 following
separation (approximately 2 hrs. after plating) were fixed
in 1% formaldehyde (ACS grade, Fisher Scientific,
Pittsburgh, PA) in phosphate-buffered saline (PBS) for 30
min., rinsed 3x in PBS, then incubated for 30 min. in 1/50
dilution of fluorescein-goat anti-mouse IgM (FITC-GAM IgM;
Boehringer Mannheim Biochemicals, Indianapolis, IN) in PBS
with 5% normal goat serum. Cells assayed for cell surface
A2B5 after one day in culture were incubated live in 1/25
A2B5-conditioned hybridoma medium +10% HI-FBS on ice and
then processed as above. Plating efficiencies of cells were
determined retrospectively from Ektachrome slides by