The wells containing bilayers were washed with RPMI culture media and warmed to 37°C for 30 min prior to adding the cells. Cells were added to the bilayer at a density of 250,000 cells/well for 4–5 min at 37°C and fixed using 4% PFA in PBS warmed to 37°C. Fixation of cells was done for 15 min at 37°C. Prior to immunostaining, cells were permeabilized with Triton X-100 (Sigma-Aldrich, T8787) at 0.1% for 5 min at room temperature and washed with PBS. The cells were blocked with 5% BSA in PBS for 1 h at room temperature.
Staining of the cells was performed sequentially with primary labelled antibodies against l CD3ζ conjugated to Alexa Flour 647 (Abcam, 197037) and pCD3ζ (pY142) conjugated to CF568. Staining was done in 5% BSA in PBS at a concentration of 10 μg/ml for both antibodies for 30 min at room temperature in the dark, then washed with PBS. Fiducials in the form of TetraSpeck Microspheres (Thermo Fisher Scientific, T7279) were added to each well for 15 min then washed with PBS.
Staining of the cells was performed sequentially with primary labelled antibodies against l CD3ζ conjugated to Alexa Flour 647 (Abcam, 197037) and pCD3ζ (pY142) conjugated to CF568. Staining was done in 5% BSA in PBS at a concentration of 10 μg/ml for both antibodies for 30 min at room temperature in the dark, then washed with PBS. Fiducials in the form of TetraSpeck Microspheres (Thermo Fisher Scientific, T7279) were added to each well for 15 min then washed with PBS.