To assess the monocytic differentiation state of THP-1 and KO52 cells, brilliant violet 421 (BV421)-conjugated monoclonal antibodies specific for human CD11b (ICRF44; BioLegend, USA) and CD14 (HCD14; BioLegend, USA) were used. Flow cytometry analysis was performed using the FlowJo software (BD Biosciences, USA).
Hcd14
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Lab products found in correlation
Hcd56, by BioLegend (2 mentions)
Icrf44, by BioLegend (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Macsxpress whole blood neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
Apc cy7, by BioLegend (1 mentions)
Astrios eq sorter, by Beckman Coulter (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Hit8a, by BioLegend (1 mentions)
Influx cell sorter, by BD (1 mentions)
Dcn228, by Miltenyi Biotec (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
M1 70, by Miltenyi Biotec (1 mentions)
Hi149, by Miltenyi Biotec (1 mentions)
Rea968, by Miltenyi Biotec (1 mentions)
Diva software, by BD (1 mentions)
Live dead yellow fluorescent dye, by Thermo Fisher Scientific (1 mentions)
Ncam16, by BD (1 mentions)
9 protocols using hcd14
1
Monocytic Differentiation State Analysis
2
Isolation and Stimulation of Human Neutrophils
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Neutrophils were isolated from 8 mL of human anticoagulated blood by negative selection using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi) according to the manufacturer's instruction. Isolated cells were stained for CD14 (BioLegend, HCD14), to select CD14 negative cells and CD15 (BioLegend, W6D3) and CD16 (BioLegend, 3G8) to confirm purity. Cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% FCS (Sigma), 100 IU/mL penicillin (Sigma), 100 μg/mL streptomycin, 2 mM l ‐glutamine (Sigma), 1 × NEAA, 10 mM HEPES, 500 μM 2‐ME. For stimulation, 1 million cells were seeded in a 48‐well plate and stimulated with 50 ng/mL TNF‐α for 18 h. Supernatants were collected and stored at −80°C until analyzed by ELISA.
3
Isolation of Monocyte Subsets
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For the isolation of classical and non‐classical monocytes from the peripheral human blood, PBMCs were incubated with specific antibodies against CD14 (PE, HCD14), CD16 (APC/Cy7, 3G8), CD56 (FITC, HCD56, all BioLegend), CD3 (VioGreen, REA613) and CD19 (VioBlue, LT19, both Miltenyi) and sorted on Astrios EQ Sorter (Beckman Coulter).
4
Immunophenotyping of PBMC Subsets
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Peripheral blood mononuclear cells (PBMCs) were Fc receptor blocked, labelled with fluorescent antibodies specific for: CD3 (OKT3), CD4 (OKT4), CD8 (HIT8a), CD14 (HCD14), CD19 (HIB19) and CD56 (HCD56; all antibodies were from Biolegend) and dead cells were identified by DAPI exclusion. CD4+, CD8+, CD14+, CD19+ and CD56+ fractions were collected (Influx cell sorter, BD Biosciences) directly into ice-cold FACS buffer, immediately frozen on dry ice and stored at –80°C.
5
Multiparametric Immune Cell Profiling
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Single‐cell suspensions from PBMCs were stained with mAb against human CD14 (HCD14; BioLegend), human CD206 (DCN228; Miltenyi Biotec), human/mouse CD11b (M1/70.15.11.5; Miltenyi Biotec), human CD1a (HI149; Miltenyi Biotec), human CD1a (HI149; Miltenyi Biotec), human CD86 (REA968; Miltenyi Biotec), human CCR7 (REA108; Miltenyi Biotec), human CD45 RA (T6D11; Miltenyi Biotec), CD4 (A161A1; BioLegend), and human CD103 (REA803; Miltenyi Biotec). After 20‐minutes incubation at 4°C in the dark, cells were washed and resuspended in PBS for the FACS analysis. For each test, cells were analyzed using FACSVerse Flow Cytometer and DIVA software (BD Biosciences).
6
Immune Cell Profiling by Flow Cytometry
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Single-cell suspensions were stained with Live/Dead yellow fluorescent dye (1:1,000, ThermoFisher) for 15 min at room temperature and washed with FACS buffer at 300xg for 5 min at 4oC. Blocking followed with 5 ul human FcR blocking reagent (Miltenyi Biotec) per 100 ul cell suspension for 15 min on ice and staining for blood immune cells was performed with the following anti-human antibodies for 30 min on ice: CD3 (1:54; UCHT1; Biolegend), CD3 (1:27; UCHT1; BD Biosciences), CD4 (1:54; RPA-T4; Biolegend), CD8 (1:54; SK1; Biolegend), CD11c (1:32; 3.9; Biolegend), CD14 (1:68; HCD14; Biolegend), CD14 (1:32; M5E2; BD Biosciences), CD16 (1:68; 3G8; Biolegend), CD19 (1:45; HIB19; BD Biosciences), CD19 (1:32; HIB19; Biolegend), CD33 (1:54; HIM3-4; BD Biosciences), CD45 (1:45; HI30; Biolegend), CD56 (1:27; B159; BD Biosciences), CD56 (1:32; NCAM16.2; BD Biosciences), CD66b (1:54; G10F5; Biolegend), CD123 (1:32; 6H6; Biolegend), CD127 (1:32; HIL-7R-M21; BD Biosciences), HLA-DR (1:68; L243; Biolegend) and Siglec-8 (1:21; 7C9; Biolegend). Cells were centrifuged at 300xg for 5 min at 4oC and resuspended in 1 ml FACS buffer. Data acquisition was performed on a 3 laser-FACS Aria III cell sorter (BD Biosciences) and were analyzed with FlowJo v10 software (BD Biosciences). Sorted neutrophils (40,000) were frozen at -80oC for further processing.
7
SAMHD1 Intracellular Staining in PBMCs
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Intracellular SAMHD1 staining was carried out as previously described9 (link). Briefly, PBMCs were stained for 30 min at room temperature using a mixture of anti-CD14-APC, anti-CD69-APC-Cy7, and anti-CD3-PB antibodies. After the cells were washed, they were fixed for 30 min at room temperature using BD Cytofix Fixation Buffer. Cells were permeabilized in BD Phosflow Perm Buffer III for 2 min at 4 °C in the dark and then were washed twice. Cells were then stained for 1 h with rabbit anti-human SAMHD1 polyclonal antibodies (1:100, Proteintech), followed by a mixture of anti-CD4-PE, and donkey anti-rabbit Alexa Fluor 488, antibodies (1:200, Invitrogen), for an additional 1 h. After the washes, cells were detected using a BD FACSAria™ II Flow Cytometer. The antibodies used for flow cytometric analysis were PE-CD4 (Biolegend, PRA-T4), APC-CD14 (Biolegend, HCD14), Pacific Blue-CD3 (Biolegend, UCHT1), APC-Cy7-CD69 (Biolegend, FN50), rabbit anti-human SAMHD1 pAb (Proteintech, 12586-1-AP), and Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) (Invitrogen, A-21206). Data were analyzed using FlowJo 7.6 software.
8
Granulocyte and Monocyte Immunophenotyping
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Basophils, eosinophils, neutrophils, and monocytes were included in all experiments and analyses of this study. Heparinized peripheral blood (stored at room temperature for less than 24 hr and protected from light) was lysed using 0.84% ammonium chloride. The leukocytes were stained for surface expression of various granulocyte and monocyte markers and processed using a FACSCanto II (Becton Dickinson, BD, New York, USA) with FACSDiva software for data collection, and analyzed with Kaluza software version 2.1 (Beckman Coulter, Brea, CA, USA). At least 40,000 granulocytes were acquired based on forward and side scatter properties. The gating strategy is illustrated in Figure S1 . A few changes regarding antibody clone or fluorophore were made after careful evaluation during the sample collection period. Antibodies recognizing the following antigens (clone) were used: CD14 (M5E2) BD Bioscience, California, USA or (HCD14) Biolegend, California, USA. CD10 (HI10a), CD16 (HIB19 or 3G8) and CD193 (5E8) BD Bioscience. CD177 (MEM-166 or GO25H7), Siglec-8 (7C9), and CD88 (S5/1) Biolegend. The CD88 antibody was included in the panel but not analyzed in this study. The addition of CD88 antibody did not interfere with the analysis.
9
Isolation and Functional Evaluation of Regulatory T Cells
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Tregs were sorted from SVF and peripheral blood (PB) based on positive viability and the following markers: Lin-CD4 + CD25 + CD127dim using an Aria III (BD) FACS sorter. Cells were stained with antibodies against CD14 (HCD14; Catalog #325604), CD15 (W6D3; #323004), CD19 (HIB19; #302206), CD20 (2H7; #302304), CD56 (HCD56; #318304); TIGIT (A15153G; #372716); CD25 (M-A251; #356110); CD8 (SK1; #344724); CD127 (A019D5; #351332); CTLA-4 (BNI3; #369609); OX-40 (Ber-ACT35; #350012); PD-1 (EH12.2H7; #329949); CD4 (RPA-T4; #300530); CD3 (OKT3; #317330); Zombie Yellow Viability Stain all from BioLegend, with validation per the manufacturer’s website (https://www.biolegend.com/en-us/quality/quality-assurance-certificates ). Additionally, cells were stained with Foxp3 (PCH101; #14-4776-82) from Invitrogen. Gating was determined based on staining antibody comparison to isotype control antibody.
To assess Treg function, previously separated PBMCs from the same patient were labeled with CellTrace CFSE (Invitrogen) and then cultured in the absence or presence of isolated blood or VAT Tregs (2 PBMC:1 Treg). Cells were cultured in a 96-well round bottom plate at 10,000 cells/well in the presence of one Treg Inspector Bead (Miltenyi) per 2 cells. After 4 days of culture, cells were stained for CD3 and viability and flow performed on an Aurora (Cytek).
To assess Treg function, previously separated PBMCs from the same patient were labeled with CellTrace CFSE (Invitrogen) and then cultured in the absence or presence of isolated blood or VAT Tregs (2 PBMC:1 Treg). Cells were cultured in a 96-well round bottom plate at 10,000 cells/well in the presence of one Treg Inspector Bead (Miltenyi) per 2 cells. After 4 days of culture, cells were stained for CD3 and viability and flow performed on an Aurora (Cytek).
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