Anti cd16 32 2.4g2

Single cells released from murine tumour tissue using enzyme dispersion, or crushed from a spleen of a 4T1 tumour-bearing mouse, were pelleted by centrifugation at 500 × g and red blood cells (RBC) were lysed using RBC lysis buffer (eBioscience) according to the manufacturer’s protocol. Human peripheral blood mononuclear cells (PBMCs) were obtained from anonymised human buffy coats as supplied by the NHS Blood and Transplant (London, UK). Buffy coats were diluted 1:2 with calcium and magnesium-free PBS (Gibco), then the PBMC fraction was isolated using a Lymphoprep (Axis-Shield) density gradient spun at 800 × g for 30 min at RT. Cells were pelleted and resuspended in PBS containing 2 mM EDTA, 1% FCS. All subsequent steps were performed on ice for 30 min unless otherwise stated. Murine Fc receptors were blocked using 5 μg/ml anti-CD16/32 (2.4G2, Tonbo Biosciences) prior to staining with F4/80 PE (BM8; to isolate macrophages) or Ly6C PE (HK1.4; to isolate monocytes) at 1 µg/ml, followed by anti-PE MicroBeads (Miltenyi Biotec). CD14⁺ human monocytes were incubated directly with anti-human CD14 MicroBeads (Miltenyi Biotec). Both human and murine cells were subsequently isolated using a MidiMacs separator and LS columns (Miltenyi Biotec) according to the manufacturer’s protocol.

Single-cell suspensions were prepared from the bone marrow of uninfected WT mice and intravaginal WT HSV-2 infected vaginal tissues of Oasl1^+/− or Oasl1^−/− mice. To obtain FACS-sorted Ly6C^high monocytes and CD4⁺ T cells, cells were stained with Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70), and CD45.2 (104) (BD Biosciences); CD3ε (145-2C11) and CD4 (GK1.5) (Tonbo biosciences) after incubating for 15 min on ice in the presence of anti-CD16/32 (2.4G2, Tonbo biosciences) antibody to block Fc receptors. Live cells were gated on the basis of DAPI exclusion. Stained cells were sorted by FACS Aria (BD Biosciences). Sorted vaginal Ly6C^high monocytes (CD45.2⁺Ly6G^-Ly6C^highCD11b⁺) and CD4⁺ T cells (CD45.2⁺CD3ε⁺CD4⁺) were more than 90% pure as assessed by post-sort analyses.