Alexa fluor phalloidin conjugates

T-cell samples were fixed with cold Dulbecco’s modified Eagle medium (DMEM) with 4% paraformaldehyde (PFA), followed by 0.1% Triton X-100 in 1% bovine serum albumin (BSA) PBS. F-actin was stained with fluorescent phalloidin (Alexa Fluor phalloidin conjugates, Thermo Fisher Scientific; 10 U/mL) or SiR-Actin (Cytoskeleton, Inc.; 1 µM), after PFA fixation for 1 h in 1% BSA PBS. Chromatin was labeled with 1 : 1000 Hoechst solution (Tocris, USA) and MTs were stained with either Alexa Fluor-conjugated rat anti-tubulin monoclonal antibody (mAb) or an unlabeled version of the same mAb clone YL1/2 (AbCam; 5 μg/mL in 1% BSA PBS, 1 h incubation). All Alexa Fluor fluorescent secondary antibody (Thermo Fisher) labelings were performed at their final concentration of 5 µg/mL for 1 h in 1% BSA PBS.

Stock solutions of (-)-blebbistatin (Sigma-Aldrich) and FAK inhibitor 14 (Tocris) were prepared 50 mM in DMSO. Before addition to cells, we incubated the solutions for 20 min in a 37 °C water bath to dissolve the drug completely, and then filtered the solutions. Controls were incubated with corresponding amounts of DMSO. After the treatment, we fixed the cells with cold DMEM with 4% PFA. We stained F-actin with Alexa Fluor phalloidin conjugates (ThermoFisher Scientific; 10 U/mL in 1% BSA PBS) and chromatin with 1:1000 Hoechst solution (Tocris).