Immunophenotypic Analysis of Cell Viability

After cells were counted, and 2x10⁶ cells per sample were stained with Aqua Live/Dead viability dye (Life Technologies) according the manufacturer’s instructions. Cells were then incubated in blocking solution containing 5% normal mouse serum, 5% normal rat serum, and 1% FcBlock (eBiosciences, San Diego, CA) in PBS and then stained with a standard panel of immunophenotyping antibodies (See S2 Table for a list of antibodies, clones, fluorochromes, manufacturers, and concentrations) for 30 minutes at room temperature. After staining, cells were washed and fixed with 0.4% paraformaldehyde in PBS. Data was acquired with a BD LSRII flow cytometer using BD FACSDiva software (BD Bioscience). Compensation was performed on the BD LSRII flow cytometer at the beginning of each experiment. Data were analyzed using Flowjo v10. Cell sorting for cytospins was performed on a BD Aria II. The collected cells were stained with a Jenner-Giemsa Stain Kit (ENG Scientific Inc, Clifton, NJ) and examined by light microscopy.