Cd45 bv786

Lymphoid, myeloid, and erythroid differentiation potential was determined using FACS analysis at 1 week post DOX induction. In all, 100% growth was obtained from all wells seeded with 300 targeted or mock-treated cells. Media were removed from all positive wells and cells were washed in 1× PBS. Cells were re-suspended in 50 μl MACS buffer (1× PBS, 2% FBS, 2 mM EDTA), blocked for nonspecific binding (5% vol/vol human FcR blocking reagent, Miltenyi, cat no. 130-059-901), stained for live dead discrimination using Live/Dead blue dead cell staining kit for UV (ThermoFisher Scientific, cat no. L23105) and stained (30 min, 4 °C dark) using CD3 PerCP/Cy5.5 (HiT3A, BioLegend), CD4 BV650 (OKT4, BioLegend), CD8 APC (HiT8a, BioLegend), CD11c BV605 (3.9, BioLegend), CD14 BV510 (M5E2, BioLegend), CD19 FITC (HIB19, BioLegend), CD33 AF-300 (WM53, BD Pharmingen), CD45 BV786 (BD Pharmingen), CD56 PE (MEM-188 BioLegend), CD235a PE-Cy7 (HI264, BioLegend), and CD271 (tNGFR) CF-594 (C40-1457, BD Horizon).

All experiments and assays were performed on freshly isolated samples. Isolated PBMCs were incubated with directly conjugated fluorescent antibodies for 30 min at 4 °C. The cells were washed before flow cytometry analysis. Monocytes were separated from other cells by gating on CD3/15/19⁻ cells combined with forward scatter (FSC)/ side scatter (SSC) characteristics and CD45 expression. The gating strategy used is shown in Fig. S1. Antibodies used included anti-human CD160-Alexa Fluor 488, CD4-APC-Fire750, CD8-BV510, HLA-DR-Alexa Fluor 700, CD14-APC, PD-1-PE, 2B4-PE-CF594, CD16-BV711, TIM-3-BV650, CD200R-PE, BTLA-BV650, CD45-BV786 (BD Biosciences, San Diego, CA, USA), CX3CR1-BV421, CD3-PerCP-Cy5.5, CD15-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD29-Alexa Fluor 488, CD62L-BV650, CD11b-BV605, CCR2-PE (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, and LAG-3-APC (eBioscience, San Diego, CA, USA), along with the corresponding isotype controls. BD Trucount Tubes (BD Biosciences), combined with specific antibodies (CD45/3/4/8 cocktail; BD Biosciences), were used to determine the absolute counts of leukocytes in the blood with flow cytometry according to the manufacturer’s instructions. The absolute numbers (cells per microliter) of leukocytes and T cells were determined by comparing cellular and bead events.

Monocyte subsets at fasting and postprandial time points were analyzed using 100 µl of whole blood collected into EDTA-treated tubes mixed with pre-titrated volumes of the following antibodies in BD Brilliant stain buffer (catalog # 563,794 BD Biosciences): CD45-BV786 (catalog # 563,716 BD Biosciences), CD91-PE (catalog # 550,497 BD Biosciences), CD14-BUV395 (catalog # 563,561 BD Biosciences), and CD16-BV421 (catalog # 562,878 BD Biosciences). Following a 20-min incubation at room temperature 1X BD FACS Lysing Solution (catalog# 349,202 BD Biosciences) was added to whole blood/antibody mixture and incubated at room temperature for an additional 10 min. Cells were washed twice with cold wash/stain buffer (containing 0.1% BSA (w/v), 0.05% NaN3 (w/v) in PBS) then analyzed using an LSR Fortessa flow cytometer (BD Biosciences) configured with blue (488 nm), red (640 nm), violet (405 nm) and UV lasers (355 nm). Data were collected using FACSDiva and analyzed using FlowJo version 10.6.1 software (BD Biosciences).

PBMCs were cultured in RPMI-1640 media (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), with or without LPS (100 ng/mL, STEMCELL Technologies, Vancouver, Canada) and Golgiplug (BD Biosciences, San Diego, CA, USA) for 3 h. The cells were surface-stained with CD45-BV786, CD14-Alexa Fluor 700, CD16-BV711, HLA-DR-APC-H7, TIM-3-BB515 (BD Biosciences, San Diego, CA, USA), and TIGIT-PE-Cy7 (eBioscience, San Diego, CA, USA), and intracellularly stained with antibodies against IL-10-APC, IL-1β-Pacific blue, TNF-α-BV650 (BD Biosciences), IL-6-PE (eBioscience), GM-CSF-PE-CF594 (BioLegend), and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).