Anti cd16 32 93

Cells were resuspended in PBS containing 2% FCS (v/v), pretreated with anti-CD16/32 (93; BioLegend) and then stained with optimal amounts of FITC-conjugated anti-CD45.1, PerCP-Cy5.5-conjugated anti-CD4 and PE-conjugated anti-CD3 (all Biolegend). Cells were analyzed on FACSCalibur (BD Biosciences) or Attune NxT Flow Cytometer (Thermo Fisher Scientific). Data analysis was performed using FlowJo software (Tree Star).

Mice were humanely euthanized by exposure to CO2. Blood was collected from mice via cardiac puncture into K3EDTA tubes and subjected to RBC lysis (eBioscience). Cells were pelleted by centrifugation at 350 X G for 10 minutes at 4°C and washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Next, cells were stained in wash buffer for an additional 20 minutes with the antibodies listed in the Key Resource Table. Stained cells were washed twice and strained through a 30 m strainer, then subjected to cell sorting using a Beckman Coulter MoFlo Astrios EQ configured with 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers. Each cell population was hierarchically gated using Beckman Coulter Summit software. Ly6C^Hi monocytes were defined asCD45^PosCD11b^HiCD115^PosCD19^NegCD90.2^NegLy6G^NegLy6C^Hi. Ly6C^Hi monocytes were further restricted to single particles by comparing height and area side scatter pulses, and dead cells were excluded by detecting the integration of the live/dead dye (Zombie NIR).