Amnis memory phenotyping expt

After overnight recovery,<b> </b>purified T cells were stained with CD4 Alexa Fluor 647, CD8 APC-H7, CCR7 BB515 and CD45RA PerCP-Cy5.5 in FACS buffer + 1% Fc block on ice for 30 minutes. Target cells were stained with CD20 BV711. T cells and target cells were washed twice in FACS buffer after mAb staining, resuspended at 2x10⁶ cells/ml in TCM, and co-incubated at a ratio of 1:1 +/- blinatumomab (10 ng/ml) for 1 hour at 37°C in a 5% CO₂ incubator. The samples were vortexed vigorously for 10 seconds and fixed by the addition of an equal volume of PBS containing 4% paraformaldehyde. After 10 minutes fixation at RT, the samples were transferred to 1.5 ml microfuge tubes and spun at 1000 rpm for 10 minutes at RT. The supernatants were removed, and the sample resuspended in 20 µl of FACS buffer. Samples were vortexed and data collected on a two camera Amnis ImageStream^X Mk II (Cytek, USA) using brightfield, 405, 488, 561, and 642 nm laser lines, and 40X/0.75NA objective. Instrument calibration and testing was performed daily. 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. Image analysis was performed with IDEAS software (Cytek, USA).