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Fig. 5. uPAR is required for keratinocyte adhesion and migration in vitro. (A) Wt and uPAR-KO primary keratinocytes were seeded on ColIV-, FN-, VN- or LN5-coated 24-well plates, or in the absence of any exogenous substrate. After 8 hours, the number of adherent cells was quantitated by staining with 0.1% crystal violet in water. Values are expressed as absorbance at 650 nm and the mean ± s.d. of triplicate samples is reported. (B) Wt and uPAR-KO primary keratinocytes were seeded for 12 hours in the absence of an exogenous substrate. Representative phase-contrast images (x20) of deposited cells are shown. (C) Wt and uPAR-KO keratinocytes were seeded on LN5- or ColIV-coated dishes and grown to confluency. Cell surfaces were scraped with a pipette tip in either a single stripe or a grid pattern. The wounds were incubated in S-MEM containing 1% chelexed FCS and photographed at various times, as indicated, with the indentation marks aligned. The lines indicate the wound edge at the start of the experiment (t=0). (D) Haptotaxis of wt and uPAR-KO keratinocytes was analyzed by 48-well Boyden chamber assay in S-MEM 1% chelexed serum through FN-, ColIV-, VN- or LN5-coated 8-µm-pore-size polycarbonate filters at 37°C for 6 hours. Results, expressed as the mean number of migrated cells ± s.d. from triplicate samples, are representative of at least three experiments.
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