Quantification of the effect of CD28 expression on cell surface spreading and tyrosine phosphorylation.

<p>The original images of the experiment of Fig. 2 were quantified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s009" target="_blank">Macro S1</a>) and the values were normalized to the mean value of the measured property within that image. Normalized values of experiments with inverted stamp and overlay configurations were pooled. The graphs show the mean ± SEM. <i>A-C</i>) Cells stimulated with stripes containing αCD3 and stripes containing αCD28. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>B & C</i>) in total counting 1010 CD28 low and 127 CD28 high cells). <i>D-F</i>) Cells stimulated with stripes containing αCD3 and stripes containing unspecific IgG2a only. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>D & E</i>) in total counting 921 CD28 low and 97 CD28 high cells). <i>G-I</i>) Cells stimulated with stripes containing unspecific IgG2a only and stripes containing αCD28. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>F & G</i>) in total counting 1006 CD28 low and 165 CD28 high cells). <i>A, D & G</i>) The background-corrected, αphosphotyrosine intensity per surface area. Corrected model p-values were determined by two-way factorial ANOVAs in which no interaction terms were included. <i>B, E & H</i>) The contact surface area per cell. Two-sample T-tests were used to generate the p-values. C, <i>F & I</i>) The integrated, background-corrected, αphosphotyrosine intensity per cell (Two-sample T-tests).