Hu fcr binding inhibitor

RPMI 1640 was obtained from Corning Technologies. Antibiotics were
purchased from Sigma. FBS purchased from Hyclone. Dead cells were excluded using
Invitrogen Fixable LIVE/DEAD in Near-IR, Yellow, or Violet. The following
antibodies were used for flow cytometry or immunofluorescence staining: from
BioLegend: anti-VISTA (clone MH5A), anti-CD45 (30-F11), anti-CD11b (M1/70),
anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-Ly6C (HK1.4), anti-Ly6G
(IA8), anti-Gr1 (RB6–8C5), anti-F4/80 (BM8), and Armenian Hamster IgG
Isotype control (HTK888); from eBioscience: anti-CD11c (N418), anti-CD16/CD32
(clone 93), and anti-FoxP3 (FJK-16s); anti- Armenian Hamster IgG (Jackson
ImmunoResearch), and anti-VISTA (clone 13F3, made in-house). Antibodies for flow
cytometry staining of human PBMCs: Hu FcR Binding Inhibitor (eBioscience),
anti-VISTA (GG8, made in-house), anti-CD14 (clone TÜK4, Miltenyi), and
from BioLegend: anti-CD11b (M1/70), anti-CD33 (WM-53), anti-HLA-DR (L243),
anti-CD3 (SK7), anti-CD19 (HIB19), and mouse IgG₁ κ isotype
control (MOPC-21). For blocking experiments, antibodies used were: anti-VISTA
(clone 13F3, made in-house) and Armenian Hamster IgG1 Isotype Control (clone
PIP, BioXCell).

Cells were treated with 0.25% Trypsin-EDTA (1X, Gibco) for 3m and
neutralized with equivalent media prior to scraping. Cells were washed with 2%
FBS PBS (400xg, 5m, 4°C), incubated (15m, 4°C) with Hu FcR Binding
Inhibitor (14–9161-73, eBioscience), washed, treated with antibodies
(30m, 4°C) prior to treatment with 2% methanol free formaldehyde (04018,
Polysciences) in PBS (15m, 23°C) and suspension in PBS. A total of 30,000
events were assessed on a Fortessa flow cytometer (Becton–Dickinson).
Data were analyzed with FlowJo data analysis software (TreeStar, Ashland, OR).
Antibodies included PerCP/Cy5.5-CD38 (356614), BV510-CD40 (334330), APC-CD80
(305220), PE/Cy7-PD-L1 (329718), from Biolegend.