15
labelled were incubated in 200 jig/ml dye solution for 30
min. according to Honig and Hume (1986). DiO stained cells
were viewed on an epifluorescent microscope with
fluorescein optics and DiO fluorescence was not visible
with rhodamine optics. Labelling efficiency with DiO was
nearly 100%.
For calculated incomplete separations, part of the
dissociated cells to be separated were incubated in A2B5 as
in a normal separation followed by incubation in the dye
DiO. These cells were then mixed with dissociated cells
that were not incubated in either of these in some ratio
(Fig. 2-6). This mix was then mixed with microspheres and
separated in the magnetic column as above. Eluted unbound
A2B5(-) cells were plated on polyornithine coverslips as
described above. After one day in culture, these cultures
along with control unseparated cultures were immunostained
live with A2B5 as described above. A rhodamine-goat anti
mouse IgM (Fisher) secondary antibody was used to
discriminate the DiO staining. The percent of the DiO(+)
cells that were also A2B5(+) were scored.
In experiments where separated cells were remixed, the
separation was carried out as normal (complete) and the
bead-bound cell fraction was recovered via trypsinization
of the beads (Fig.2-7). Eluted A2B5(-) cells were meanwhile
incubated in DiO, and then the two separated cell fractions
were remixed in even proportions. After one day in culture,