Fc receptor blocker

PBMCs were extracted from healthy subjects. PBMC samples were divided into the CD47 isotype control group, anti-CD47 antibody group, LPS group, and anti-CD47 antibody + LPS group. The cells were cultured in RPMI-1640 medium containing 10% FBS. According to the grouping, 1 μg/mL of isotype control (#14-4321-85, eBioscience), 1 μg/mL of anti-CD47 antibody (#16-0471-81, eBioscience), 3 μg/mL of LPS (#abs47014848, absin), 1 μg/mL of anti-CD47 antibody (#16-0471-81, eBioscience) + 3 μg/mL of LPS (#abs47014848, absin) was added to PBMC (10⁵ cells/mL), respectively. After mixing, they were cultured in 37°C, 5% CO₂ incubator for 5 h. Cells were collected and treated with Fixable Viability Dye eF780(#65-0865, eBioscience, USA) and FC receptor blockers (#564765, BD Biosciences, Germany). After washing, 5 μL of fluorescent dye CD14-BV510 (#563079, BD Biosciences) was added and incubated at 4°C for 30 min in the dark. Washing, an intracellular staining antibody (TNF-α-PE-c) (#557647, BD Biosciences) was added. Washing again, the cells were detected by CytoFlex V5-B5-R3 Flow cytometer (No: 38385, Beckman USA) Flow cytometer.

After treated with Fixable Viability Dye eF780(#65-0865, eBioscience, USA) and FC receptor blockers (#564765, BD Biosciences, Germany), PBMCs (10⁵ cells/mL) were incubated for 30 min at 4°C in the dark with 5ul of fluorescent dyes CD3-Percp-Cy5.5 (#560835, BD Biosciences), CD19-BV421 (#562440, BD Biosciences), CD14-BV510 (#563079, BD Biosciences), CD56-BV650(#564057, BD Biosciences), SIRPα-APC (#17-1729-42, eBioscience), 20ul of CD16-PE (#555407, BD Biosciences) or CD47-FITC (#556045, BD Biosciences). After washing, the cells were detected by CytoFlex V5-B5-R3 Flow cytometer (No: 38385, Beckman USA). The expressions of CD47 and SIRPα in CD3⁺ T cells, CD19⁺ B cells, CD56⁺ NK cells or CD14⁺ monocytes in PBMC were analysed using flowjo 6.2 software. The gate-drawing strategies for CD3⁺ T cells, CD19⁺ B cells, CD56⁺ NK cells and CD14⁺ monocytes were shown in Figure 1.

To characterize the MDSC populations in freshly isolated PBMCs, a range of antibodies were used, including HLA-DR, CD33, CD14, CD15, CD3, CD19, and CD56 (BioLegend, San Diego, CA, USA). Moreover, antibodies against the following surface antigens were used to evaluate MDSC differentiation: CD86, CD11c, and CD206. All stained samples and their respective isotype controls were incubated in PBS containing 0.5% bovine serum albumin (BSA) and FC-receptor blockers (BD Biosciences, San Jose, CA) to restrict non-specific binding sites. The results obtained were evaluated by a MACSQuant 10 Analyser (MiltenyiBiotec, Germany). A minimum of 10,000 live events per sample were acquired for analysis. Briefly, following the initial forward versus side scatter (FSC vs. SSC) discrimination, the gate was set on HLA-DR⁻/CD33⁺ cells. Next, we gated on the subpopulations defined as MDSC, including G-MDSC (HLA-DR^-CD33⁺CD15⁺), M-MDSC (HLA-DR^-CD33⁺CD14⁺) cells, and their combinations. Moreover, all data analyses were carried out using FlowJo software (Tree Star, Ashland, OR).

Cultured cells were incubated for 20 min at 4 °C with the following antibodies mouse IgG1 PE-conjugated TRAIL clone RIK-2 (1/100) (BD Bioscience, San Jose, CA), mouse IgG1 APC-conjugated BDCA-4 clone REA380 (1/100), mouse IgG2a FITC-CD123 clone AC145 (1/100) (Miltenyi, Bergisch Gladbach, Germany), mouse IgG2a FITC-HLADR clone L243 (1/200), mouse IgG2a PercP-cy5.5-CCR7 clone G043H7 (1/50), mouse IgG1 APC-CD40 clone 5C3 (1/50), mouse IgG1 BV421-CD80 clone 2D10 (1/50), mouse IgG2b AF488-CD86 clone IT2.2 (1/50), mouse IgG1 PE-CXCR4 clone 12G5 (1/100) (Biolegend, San Diego, CA) or with appropriate isotype-matched control antibodies (5 μg ml⁻¹ each) in PBS containing 2% foetal bovine serum (Sigma, Saint Louis, MO) and FC-receptor blockers (BD Biosciences, San Jose, CA). Flow cytometry analysis was performed on a BD Canto II or LSR II flow cytometer using flow cytometry Diva software (BD Biosciences, San Jose, CA). FlowJo software (Treestar, Ashland, OR) was used to analyse data.

Cytospins of single cell suspensions were fixed with 4% formaldehyde, permeabilized with 0.5% saponin, and blocked with 3% BSA and Fc receptor Blocker (BD BioSciences). Then cells were stained with anti-γH2AX (Millipore) for overnight and detected by antibodies labeled with DAPI (4′,6-diamidino-2-phenylindole) and Alexa Fluor 488. Images were detected with Nikon ECLIPSE TE2000 and NIS-Elements software version 2.30 and Leica DFC300 FX.

In general, cell surface staining was performed in ice-cold PBS containing 0.5% BSA for 30 min at 4 °C. Non-specific staining was blocked using Fc receptor blocker (BD Biosciences). Dead cells were excluded using the fixable viability dye eFluor780 (eBioscience, USA). Transcription factor staining was performed after using the Foxp3 Fixation and Permeabilization Kit (eBioscience). Surface molecule staining was performed before fixation. For intracellular cytokine staining, cells were stimulated in vitro for 6 h with Leukocyte Activation Cocktail (BD Biosciences) and stained according to the manufacturer's instructions. The following antibodies were used: live/dead (FVS780), anti-CD45 (HI30, 13/2.3), anti-CD4(RM4.5), anti-CXCR2 (SA0446), anti-CD3ε (152,303), anti-CD8α (53–6.7), anti-CD11c (B-ly6), anti-CD86 (CL-1), anti-G4S linker (GS-ARAP25), anti-Gr-1 (RB6-8C), anti-CD206 (C068C2), anti-CD62L (MEIL-14), anti-CD44 (IM-7)and isotype controls (MOPC-21,27–35,X40,R35-95). All antibodies were purchased from BD Bioscience and Biolegend (San Diego, USA).

Tumors were excised, minced and further dissociated at 37°C with collagenase IV (Worthington Biochemicals) in DMEM, 0.5% bovine serum albumin, 1X pen/strep/fungi, and 2 µL/mL DNAse (Ambion) by shaking at 180 rpm for 30–60 min. Dissociation was complete after final trituration using a 5 mL pipette and then filtering the material through 70 uM and then 40 uM filters. Single cells were stained with anti-CD31 (BD Biosciences) or an isotype control antibody for 30 min. at 4°C. After washing, a secondary APC-conjugated antibody was added for 30 min. at 4°C. Cells were then washed with PBS and fixed with Cytofix fixation buffer (BD Biosciences). For analysis of CD133 expression on cultured cells, cells were diluted in FACS buffer of PBS containing 2% FBS and 1 mM EDTA. They were preincubated with Fc receptor blocker (BD Biosciences) for 20 min at 4°C and then washed thrice. APC-conjugated anti-Prominin-1 antibody or the isotype control (Multenyi Biotec) were diluted in FACS buffer at 1∶200, and added to the cells for 30 min incubation at 4°C. Cells were then washed thrice in cold PBS and fixed with fixation buffer (BD Biosciences). All flow cytometric analysis was performed on a Becton-Dickinson LSR analyzer (BD Biosciences) and analyzed using FlowJo (Tree Star, Inc.) software.