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Fig. 2. Phenotypic analysis of CMP-FLEC isolates: purity of CMP-FLEC isolated
with micromagnetic beads was assessed by IIF analysis of cytospins or by FACS
analysis. (A) Cytospin of an unfractionated ED16 total fetal liver
isolate labeled with MAb OC.2 (isolates A and F,
Table 1). Only subpopulations
of cells were strongly positive for OC.2 (arrows). (B) Cytospin of MAb
OC.2 reactive cells recovered from ED16 fetal liver isolate with micromagnetic
beads (isolate B, Table 1).
Greater than 95% of cells were strongly positive for OC.2. (C) Cytospin
of an unfractionated ED18/19 total fetal liver isolate labeled with MAb OC.10
(isolate O, Table 1). Only a
subpopulation of cells showed positive staining (arrow). (D) Cytospin
of MAb OC.10 reactive cells recovered with micromagnetic beads from ED18/19
fetal liver cell isolate (isolate G, Table
1). All of the cells were positive for OC.10. (E,F)
FACS analysis of MAb BD.2/MAb OC.5-positive and -negative cell populations
after micromagnetic bead fractionation (isolates P and Q,
Fig. 1). The black shaded peak
represents background levels of fluorescence exhibited by cells labeled with a
cocktail of MAb BD.2 (IgG isotype) and MAb OC.5 (IgM isotype) followed by a
cocktail of nonspecific secondary antibodies (FITC-conjugated rabbit anti goat
IgG and rabbit anti goat IgM antibodies). Unshaded peak shows the level of
fluorescence following sequential incubation with a mixture of MAbs BD.2 and
OC.5, and a mixture of goat anti-mouse IgG and goat anti-mouse IgM antibodies.
More than 95% of the cells in the positive fraction labeled with appropriate
secondary antibodies displayed fluorescence above background levels (E,
isolate P, Table 1). By
comparison, fewer than 2% of cells remaining after depletion of positive cells
showed fluorescence levels greater than the negative control (F, isolate Q,
Fig. 1). Scale bars: 50
µm.
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