Fluorescence-Activated Cell Sorting (FACS)

To obtain all necessary cell populations of interest, five-way fluorescence-activated cell sorting was performed using a Duke Human Vaccine Institute (DHVI) BD Influx instrument with a 70 μm nozzle, producing a drop size of ~1.5 nL/cell. Cold blocks were used to maintain the sort sample and collection samples at 4°C, and the sample was collected into RNase-free 1.5 mL tubes containing 500 μL of collection buffer (1% BSA, 4M NaCl in RNase-free PBS) to account for dilution. A maximum of 500 μL of cells were sorting into each tube to prevent dilution below 2M NaCl. When the Influx instrument was not available, 4-way sorting was performed using BD Aria II cell sorter in a similar manner. In this case, the negative population was isolated first (pop A), followed by four-way sorting to obtain pop. B-E. Immunophenotyping experiments were performed using a BD LSRFortessa, X20, a BD Aria II, or a Cytek Northern Lights spectral flow cytometer. All instruments used in these experiments undergo routine preventative maintenance and quality control to ensure optimal functioning.