BV421 Rat Anti-Mouse Siglec-F

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell)	Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences).

Protocol tips
Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell)
Downstream tips
Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences).

Publication protocol

Single cell suspensions were stained with fluorescently conjugated antibodies in a 1:100 dilution unless otherwise noted. Cells were stained with LIVE/DEAD™ Aqua or Blue (Invitrogen), blocked with 4μg/ml anti-CD16/32 (2.4G2; Bioxcell) and the stained with anti-CD11b eFluor450 (M1/70; Biolegend), anti-CD11b BV650 (M1/70; Biolegend), anti-CD11b AF700 (M1/70; Biolegend) anti-MHCII APC-Cy7 (M5/114.15.2; Biolegend), CD45.1 FITC (A20; Biolegend), CD45.1 PerCP/Cy5.5 (A20; Biolegend), CD45.2 FITC (104; Biolegend), CD45.2 PerCP/Cy5.5 (104; Biolegend), F4/80 PECy7 (BM8; Biolegend), CD206 APC (C068C2; Biolegend), Siglec-F BV421 (E50-2440; BD Biosciences), Siglec-F PE (E50-2440; BD Biosciences), PD-L2 PE (TY25; Biolegend), PD-L2 PerCP-Vio700 (MIH-37; Miltenyi Biotec) CD3 PE (17A2; Biolegend), CD19 PE (6D5; Biolegend), CD49b PE (DX5; Biolegend). For EdU labeling, mice were pulsed with 0.5 mg of EdU for 3h prior to sacrifice and single cell suspensions were surface stained, fixed in 2% PFA for 10 min at RT, permeabilized, and stained for EdU (Invitrogen) per the manufacturer’s instructions. Samples were analyzed on an LSRII and sorted on an Aria II cell sorter (BD Biosciences). For FACS-purifying peritoneal macrophages, cells were gated on singlet, live, Dump-negative (CD3−, CD19−, DX5−, Siglec-F−) CD11b+ then subsequently gated on their AAMres (F4/80hi, CD206−, tdTomato−), AAMmono (F4/80int, CD206+, tdTomato+) or AAMconv (F4/80hi, CD206−, tdTomato+) phenotype.

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