Flow Cytometry Immunophenotyping Protocol

Antibody reagent cocktails were made in 5 ml round-bottom tubes, immediately prior to use, as specified in Table 1. One hundred microliters of cells separated (approximately 1 × 10⁶ cells) in phosphate-buffered saline (PBS) were added. Tubes were incubated for 20 minutes in the dark at room temperature. Red blood cells (RBCs) were lysed by adding 1 ml of 1X BD Pharm Lyse lysing buffer to each tube followed by 10 minutes of incubation in the dark at room temperature. Acquisition and analysis were done using a FACSCanto flow cytometry (Becton Dickinson Biosciences, San Jose, California USA). One hundred thousand events were analyzed, and an isotype-matched negative control was used with each sample [12 (link)].