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(E) Single cell colony formation.
Consistent with this observation, we also found that the capability of single cell colony formation was much stronger in LBX medium (Fig. 1E).
To define whether adipogenic potential is restricted to a specific subset of ObL cells, we used a combination of single cell colony assays and replica plating [24], [25].
Cells were selected with 10 μg/ml puromycin before single cell colony selection to identify low expression GFP FYVEHrs clones.
Isolates were purified by serially streaking a single cell colony three times on to new agar plates.
Another indication of the growth and colony formation promoting effect of an extra-3p loss was obtained from the single cell colony analysis of #3/KH39 (figure 4F).
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Positive cells were selected for GFP expression by FACS-sorting and single cell colonies were isolated by ring-cloning.
To obtain single cell colonies for clonal analyses, cells were plated out at a low density (20/cm2) for 24 hrs and maintained at 20°C as previously described [20].
Fortunately, the overall rate of external contamination observed in this study was low and true somatic mutations could be detected in single cells [13], [23] or single cell colonies [24].
After being picked with a micropipette, some single cell colonies were expanded in medium containing VEGF (Fig. 6A H) to evaluate the direct effect of this cytokine on the myogenic differentiation program of the leech myoendothelial cells.
After transfection, cells were treated with 1.5 μg/mL puromycin and single cell colonies were collected.
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