Cd16 32 pe

Individual yolk sacs were incubated in 100 μL of 0.125% Type I collagenase (Sigma) at 37°C for 15 min, diluted and pipetted 20× with 250 μL of 1× PBS/1 mM EDTA, and digested an additional 15 min. Samples were pipetted 20–30×, diluted with 250 μL of 1× PBS/1 mM EDTA, and filtered to single-cell suspension. Cells were blocked with 10% normal rat serum (15 min) and incubated with CD117 APC-eFluor 780 (eBioscience), CD41a PE-Cy7 (eBioscience), CD16/32 PE (eBioscience), and Ter119 APC (BD) antibodies (1:100) for 20 min on ice. Cells were washed, stained with 4,6-diamidino-2-phenylindole (DAPI; 5 μg/mL), and analyzed with an LSRII cytometer (BD). Data were analyzed using FlowJo X Software (BD).

Peripheral blood samples were stained with CD45 PerCP-Cy5.5/APC Cy7, B220 APC/PE Cy7, Gr1 Alexa Fluor 700, Mac1/CD11b, CD3e APC/PE, CD4 PE, and CD8a APC/PE.
Whole bone marrow cells were stained as above for mature lineages. Cells were also stained for Lineage^– Sca1⁺ Kit⁺ CD48^– CD150⁺ (LSK SLAM) with biotin-conjugated anti-mouse lineage antibodies (Ter119, B220, Gr1, CD11b, CD3e) followed by staining for streptavidin V500/eF450 (eBioscience [eF450]), c-Kit APC eF780/APC (eBioscience [APC eF780]), Sca1 PE Cy7, CD48 AF700/BV605 (Biolegend [AF700]) CD150 APC/PE (eBioscience [APC]; Fisher Scientific [PE]). Bone marrow cells were further stained with CD16/32 PE (eBioscience) and CD34 eF450 (eBioscience) to immunostain for committed progenitor populations. For mitochondrial function, cells immunostained for LSK SLAM were incubated at 37°C in 5% CO₂ for 30 min with either MitoSOX Deep Red Reagent (1 mM, Invitrogen) or tetramethylrhodamine ester (0.1 mM, Sigma Aldrich). Caspase 1 activity was determined using the FLICA assay (Corning), according to the manufacturer’s recommendations. Samples were then analyzed using a BD LSR II, BD LSR Fortessa, or BD Canto III (BD Biosciences). All antibodies were obtained from BD Biosciences, unless otherwise noted.