Igg2a clone mopc 173

Melanoma 624 wt or knockout cells were plated at equal densities and incubated overnight. Resuspended cells were incubated on ice for 1 h with the primary antibody at a concentration of 0.2 μg/well in FACS buffer (1× PBS, 0.5% bovine serum albumin, 0.05% NaN₃). The cells were then incubated for 30 min on ice with anti-mouse AlexaFluor 647 secondary antibody (Jackson ImmunoResearch). The following primary antibodies were used: anti-MICA (clone 159227, R&D Systems), anti-MICB (clone 236511, R&D Systems), anti-ULBP1 (clone 170818, R&D Systems), anti-ULBP2/5/6 (clone 165903, R&D Systems), anti-ULBP3 (clone 166514, R&D Systems), anti-B7H6 (clone 875001, R&D systems), anti-PVR (in-house developed), anti-HLA1 (W6/32), anti-Beta-2 microglobulin (β2M, clone 2M2, Biolegend), anti-Ceacam-1 (clone ASL-32, Biolegend), anti-Nectin-2 (clone TX31, Biolegend). Mouse IgG1 (clone MOPC-21, Biolegend), IgG2a (clone MOPC-173, Biolegend), and IgG2b (clone MPC-11, Biolegend) were used as an isotype control.

The mouse anti-CD162 (PSGL-1) mAb (clone KPL-1, Cat# 556052, Lot# 3225805) were purchased from BD Biosciences. The mouse IgG₁ isotype control (clone MOPC-21, Cat# 400124, Lot# B136629) and IgG_2a (clone MOPC-173, Cat# 400202, Lot# B223740) were purchased from BioLegend. The goat anti-SCARB2 pAb (Cat# AF1966, Lot# KKY0115011 [Figs 5A, 5B and 8A], KKY0118021 [S1 and S7 Figs], KKY0118031 [Figs 8B, 9B, S2B and S6], KKY0121011 [Figs 2A, 3 and 9C]), normal goat IgG (Cat# AB-108-C, Lot# ES4119121, except for Fig 9C where Lot# ES4521041 was used), and the goat anti-human IgG Fc Ab (Cat# G-102-C, Lot# WBT1519101) were purchased from R&D Systems. The rabbit anti-SCARB2 mAb (clone 12H5L1, Cat# 702770, Lot# 2110715; clone 22H6L14, Cat# 703037, Lot# 2360697), rabbit IgG isotype control (Cat# 10500C, Lot# UA276761), the mouse anti-CD63 mAb (clone MEM-259, Cat# MA1-19281, Lot# WG3317432A), and the mouse anti-CD107a (LAMP-1) mAb (clone eBioH4A3, Cat# 14-1079-80, Lot# 2440973) were purchased from Invitrogen. The mouse anti-EEA1 mAb (clone 3C10, Cat# M176-3MS, Lot# 003) was purchased from MBL.

PBMCs were isolated as above, resuspended in PBS, and Fc receptors blocked with anti-CD16/32 (BD Biosciences) on ice for 10 minutes prior to staining with anti–human CD14–FITC (Biolegend, clone HCD14) and anti–human Dectin-1–PE (Biolegend, clone 15E2), or the appropriate isotype control (Biolegend, IgG2a clone MOPC-173), on ice for 30 minutes. Stained samples were washed in PBS and acquired using a BD LSR, equipped with BD FACSDiva software. FlowJo (BD) was used for the final analysis.