H flow cytometry for SHP2 KD cells (A) and wt cells (B). The unfilled histograms show isotype controls though the filled histograms aCD3 and aCD28 labeled populations, respectively. (TIF) Figure S7 CFSE fluorescence (green) is retained by all cells following fixation, permeabilization and immunolabeling. Stamps coated with 25 mg/ml aCD3 had been employed to produce striped patterns (blue) which had been overlaid with 2.5 mg/ml aCD3 + 2.five mg/ml aCD28. Jurkat E6.1 `wild type’ cells were labeled with CFDA-SE (A) or mock labeled (B), serum starved over evening and subsequently incubated around the micropatterned surfaces for 10 minutes, fixed with three PFA and immunolabeled with aphospho-PLCc1 (grayscale). A B have been recorded with identical microscopy settings and all 3 channels are overlaid for both. For clarity, contrast and brightness have been adjusted proportionally. Scale bar 50 mm. (TIF) Figure S8 SHP2 knock down effect on phosphatidylser-Overlay of common microscopy pictures employed for evaluation. One particular field of view at 2048 6 2048 pixels. Within this case stamps coated with 25 mg/ml aCD3 have been made use of to produce a striped pattern (blue) which was overlaid with 2.five mg/ml aCD3 + two.5 mg/ml aCD28. The CFSE labeled (green) SHP2 KD Jurkat T cells are clearly distinguishable from the non-CFSE labeled wt Jurkat cells. Right after fixation with three PFA the cells have been immunolabeled with aphospho-PLCc1 (grayscale). For clarity, contrast and brightness are adjusted proportionally. Scale bar main image 50 mm; scale bar enlargement 10 mm. (TIF)Figure S3 Figure S4 Tyrosine phosphorylation on manage surfac-es. CD28-GFP transfected Jurkat ACC-282 T cells were serum starved for 6 h and after that incubated on striped surfaces for 10 minutes, fixed with 3 PFA and immunolabeled with aphosphotyrosine. Surfaces have been functionalized using stamps coated with 25 mg/ml aCD3 (A) or unspecific IgG2a only (B).1416990-09-4 web The remainder was subsequently overlaid with either five mg/ml aCD28 (A) or unspecific IgG2a only (B). Best left panels: transmission image; top ideal panels: CD28-GFP; bottom left: aphosphotyrosine; bottom suitable panels: overlay in the stamped pattern (blue) plus the aphosphotyrosine label (grayscale). To get a greater comparison no adjustments have been produced towards the contrast or brightness in the images. Scale bars 50 mm. (TIF)Figure S5 Lowered adherence and spreading of cellsine exposure. Wells of a 96-well flat bottom plate had been coated as described for the ELISA inside the Components and Strategies section. In these wells 1N105 SHP2 KD or wt Jurkat T cells had been stimulated with aCD3 aCD28 (clone CD28.94-75-7 site two; eBioscience, Frankfurt, Germany), aCD3 alone, aCD28 alone or were left unstimulated (-) for 24 (left) or 48 hours (proper) at 37uC, five CO2 and under humidified conditions.PMID:24078122 Cells had been subsequently stained together with the Annexin V-PE 7-AAD Apoptosis Detection Kit I (BD Pharmingen, Heidelberg, Germany) utilizing the suppliers protocol. Phosphatidylserine exposure was determined employing a FACS Canto flow cytometer (BD Biosciences, Heidelberg, Germany) and characterizing 1N104 cells per sample. The graph shows the percentage of annexin V unfavorable cells six SEM of 3 independent experiments. (TIF)Macro S1 Macro utilized for information extraction from imagestreated with cytochalasine D. Jurkat T cells had been serum starved overnight and have been treated with 10 mM cytochalasine D (Tocris Bioscience, Bristol, UK) ten minutes before, and through incubation on striped surfaces. Surfaces were functionalized applying stamps coated with 25 mg/ml aCD3 and overlaid with.
