Figure 2.

FACS analysis of trypsinized ESC-MEF co-culture with/without re-plating on a gelatin-coated dish. (A) Confirmation of specific signaling using fluorescent microscopy prior to FACS: GFP-positive ESCs exhibited high-intensity signal in the green emission spectrum, MEFs labeled using the anti-feeder-PE antibody emitted a specific red signal. (B) Grainy pattern of the specific anti-feeder-PE labeling, as previously observed in the immunocytochemistry data. (C) Trypsinized ESC-MEF cell suspension showed a well-delineated GFP+ cell population depicted on the right side of the plot, with the anti-feeder-PE + MEF- fraction situated in the left upper corner; this particular measurement showed the highest measured value of 9.9% of the overall cell-count. (D) Amount of MEFs contaminating the cell suspension decreased to a minimum of 0.9% after re-plating step (Note: both plots show representative maximum and minimum values acquired by FACS assessment of two individual cell-culture dishes, which was further followed by repeated measurements of additional dishes – see next) (E) Bar diagram presenting the absolute values (mean ± SD) of n = 7 untreated cell suspensions and n = 7 after re-plating on gelatin-coated dish, with a total cell loss accounting for approximately 10% of the primary cell suspension. The cell loss was statistically significant, when examined for both the entire cell suspension and GFP+ fraction – marked by *p < 0.001. (F) Additional diagram presenting %-mean and ±SD values of FACS-acquired MEF contaminations in untreated versus gelatin-treated dishes, showing a statistically significant reduction of MEF amount – marked as ^#p = 0.011. Despite the reduction, MEF contamination still accounted for 1.4 ± 0.2% of the entire cell suspension.