Figure 3

7AAD⁺ Keratinocytes show intense staining with any polyclonal primary antibody. Keratinocytes of donor C were harvested following high-density culture for 6 days, either in standard keratinocyte (iKer) or maturation medium (dKer). (A) All cells being stained with indicated rabbit pan antibodies were 7AAD⁺. Left column shows FACS plots as used by Clark et al. [15], right column contains additional information regarding viability. Within experiments, the percentage of positively stained cells was always similar with any pan-antibody. Insert shows background staining by the secondary antibody used. Even though staining intensity was lower after incubation with the secondary antibody, when compared with unstained cells (LL quadrant of the right column), the percentage of cells with signal was similar. This staining-pattern was observed for all donors. (B) When gated on 7AAD^- cells, any pan antibody stained a low percentage of keratinocytes (donor C, donor D showed similar inconclusive staining profile). (C) Almost all cells that were stained with H265, co-stained with H20. A nearly similar plot could be obtained with any pan rabbit (eg ACE) and pan goat antibody combination (eg, NOS2), and invariably involved 7AAD⁺ cells (donor C, donor D showed similar co-staining).