WASp^I294T 300.19 cells display altered contact dynamics to L-selectin ligand. (A) WASp^WT and WASp^I294T 300.19 cells were plated on either PLL or sLe^x (B) and were imaged by rapid time-lapse IRM. sLe^x was immobilized as described in supplemental Methods using glass coverslips. Images were acquired in normal growth medium at 37°C using a Zeiss Standard18 microscope with IV-FL incident light fluorescence attachment, appropriate filters for IRM acquisition, and a Zeiss 63×/1.25 NA Neofluar Antiflex oil-immersion objective and a Pulnix CCD camera (Jai Ltd) using in-house acquisition software (Matrox Video and Imaging Technology Europe Ltd). Composite images of 4 consecutive frames analyzed for adhesion contacts (using Adobe Photoshop as described in supplemental Methods) are shown with a full-field view above an enlarged individual cell. Separate scale bars are shown for original magnification and enlarged images. Stable adhesions appear as black/dark gray pixels, whereas dynamic adhesions appear light gray. WASp^I294T cells show an increase in stable adhesions compared with WASp^WT cells when adherent to L-selectin ligand (sLe^x) but not to PLL. (C) Stable adhesions were calculated for individual cells and displayed as a percentage of total adhesion pixels. Quantifications of stable adhesions are shown for PLL and sLe^x in left and right panels, respectively. WASp^I294T cells demonstrate a significant increase in stable adhesions when adherent to sLe^x but not PLL (P < .001; 2-tailed t test).