Amnis phalloidin immune synapse expt

Staining for F-actin was performed on the fixed samples from the conjugation assay. Samples were incubated at RT in the dark for 10 minutes once the fixative was added at the end of the conjugation incubation. 400 µl of FACS buffer was added and the samples spun at 524 g for 4 mins. Cells were resuspended in permeabilization buffer (0.1% Triton-X-100 in 50mM Tris-HCL, pH7.6) and incubated at RT in the dark for 10 minutes. The samples were washed in 400 µl of FACS buffer and spun at 524 g for 4 mins. Cells were resuspended in 100 µl of Phalloidin Alexa Fluor 488, diluted 1:800 in FACS buffer + human Fc block. Samples were incubated for 20 mins at RT in the dark, washed in 400 µl of FACS buffer at 524 g for 4 mins, resuspended in 400 µl FACS buffer and transferred to an Eppendorf tube and spun at 1000 rpm for 10 minutes at RT. The sample was resuspended in 20 µl of FACS buffer and acquired on an Amnis ImageStream^X Mk II cytometer. In focus cellular events were determined by gating on events with high gradient RMS values and high area vs aspect ratio from the brightfield channel, following which cell complexes were determined by co-expression of T and tumour cell line markers and synapse detection by phalloidin staining. Image analysis was performed with IDEAS software (Cytek, USA).