35 μm nylon cell strainer mesh tubes

Imaging flow cytometry analysis was carried out using an ImagesStream^X Mark I platform (Amnis-Merck-Millipore) in standard configuration, equipped with multi-magnification (20X, 40X, 60X), extended depth of field, 405 nm and 488 nm excitation lasers and a 785 nm laser for a scatter signal with standard filter sets. INSPIRE software (Amnis, USA) was used for acquisition and IDEAS software (Amnis, USA) for analysis. Prior to sample acquisition, the machine was calibrated and tested using the standard machine scripts. Cells were filtered through 35 μm nylon cell strainer mesh tubes (BD Biosciences, UK) immediately prior to acquisition and a minimum of 30,000 events per sample were acquired using the 40X objective. The cell classifier was used to set an area upper limit (AUL) of 600 units and an area lower limit (ALL) of 50 units in the brightfield channel to define cellular events, events outside this range were classed as debris and excluded from collection. All channels were collected with the laser power set to 50.0 mW for the 488 nm laser and 3.09 mW for the 785 nm scatter laser. Single stain compensation tubes for each stain used, as well as an unstained tube, were prepared alongside test samples and run for the generation of compensation matrices.