The following antibodies were used to identify cell subsets: Ly6C-FITC (AL-21, BD Biosciences), F4/80-PerCP-Cy5.5 or APC eFluor660 (BM8, eBioscience), Siglec-F-PE (E50-2440, BD Biosciences), CD11c-PE or BV711 (HL3, Biolegend), and Ly6G-PerCP eFluor710 or V450 (1A8, eBioscience or BD Biosciences). Dead cells were excluded from analyses using Fixable Viability Dye APC eFluor780 or BV506 (eBioscience).
Surface staining: Cells from all samples were adjusted to an equal concentration, and treated with anti-CD16/CD32 Fc receptor blocking antibody (clone 2.4G2) in 1x PBS (1% FBS) for 10 minutes on ice. Surface staining antibodies were then added and incubated for 15 minutes on ice. Cells were washed with 1x PBS then incubated with Fixable Viability Dye diluted in 1x PBS for 15 minutes on ice. Cells were washed, then fixed with 1% paraformaldehyde for 15 minutes on ice.
Samples were acquired using an Attune NxT Acoustic Focusing Cytometer with Attune Software or a BD FACSAria with FACSDiva Software. Analyses were performed using FlowJo v10 software (Tree Star, Inc.). Gate placement was determined using isotype, FMO, or unstained control samples. Total cell numbers of each cell subset was obtained by using the total cell counts of the compartment as described above, and multiplying by the percent of total viable cells as determined by flow cytometry.
Surface staining: Cells from all samples were adjusted to an equal concentration, and treated with anti-CD16/CD32 Fc receptor blocking antibody (clone 2.4G2) in 1x PBS (1% FBS) for 10 minutes on ice. Surface staining antibodies were then added and incubated for 15 minutes on ice. Cells were washed with 1x PBS then incubated with Fixable Viability Dye diluted in 1x PBS for 15 minutes on ice. Cells were washed, then fixed with 1% paraformaldehyde for 15 minutes on ice.
Samples were acquired using an Attune NxT Acoustic Focusing Cytometer with Attune Software or a BD FACSAria with FACSDiva Software. Analyses were performed using FlowJo v10 software (Tree Star, Inc.). Gate placement was determined using isotype, FMO, or unstained control samples. Total cell numbers of each cell subset was obtained by using the total cell counts of the compartment as described above, and multiplying by the percent of total viable cells as determined by flow cytometry.