Detection of the stimulus dependence of total tyrosine phosphorylation (<i>B</i>) and phosphoY783 PLCγ1 (<i>C</i>) in Jurkat cells and SHP2 KD cells.

<p><i>A</i>) For the side-by-side analysis of signaling in Wt and SHP2 KD Jurkat E6.1 T cells, one of the lines was labeled with the cell tracer CFSE. After overnight serum starvation the cells are pooled and incubated on micropatterned, stimulating surfaces for 10 min. Subsequently, the cells are fixed with 3% PFA, permeabilized and immunolabeled for the detection of signaling clusters. <i>B & C</i>) In the top panels, SHP2 KD cells are CFSE labeled and in the bottom panels, wt cells are labeled. Panels from left to right: transmission images; CFSE; immunofluorescence; overlay of the stamped pattern (blue) and the immunolabel (grayscale). In the overlay panels the contrast and brightness for both channels were adjusted proportionally for clarity. 12.5 µg/ml αCD3 + 12.5 µg/ml αCD28 coated stamps were used to generate a striped pattern which was overlaid with 5 µg/ml αCD3. CFSE channels were recorded with saturated signals to facilitate image processing. Scale bars 20 µm.