Figure 8.

Quantitative measurements of keratinocyte cell adhesion as determined by a novel dispase-based assay. (A) Primary keratinocytes under low calcium conditions (Low Ca) or treated with calcium for 2 or 9 h (Ca) were incubated with dispase for 35 min as described in Materials and Methods. Data are expressed as percentage of single cells released by mechanical disruption after dispase treatment versus total number of cells recovered after subsequent treatment of the same samples with trypsin. (B) A similar assay was performed with keratinocytes under low calcium conditions or incubated with calcium for 9 h (Ca) in the absence or presence of Genistein (100 μM) (Gen), Thyrphostin (100 μM) (Trph), or PP1 (1 μg/ml). Cells were preincubated with the inhibitors for 2 h before calcium treatment. (C) Keratinocytes under low calcium conditions were infected with a control adenovirus expressing the green fluorescent protein (Ad-GFP) or a virus expressing a constitutively active form of c-src (Ad-src). 24 h after infection, cells were switched to high calcium medium and incubation was continued for additional 24 h (Ca). Cells were analyzed by the dispase assay as before, together with control uninfected cells kept in either low or high calcium medium for 24 h.