Cell populations in BM-MNCs were identified with the FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA). 10 × 106 cells were washed in PBA (0.5% BSA in PBS) after thawing, blocked with Fc-block (Miltenyi Biotec, Bergisch Gladbach, Germany), stained for 45 min at 4 °C in the dark with the monoclonal antibodies (CD34 BV421 Biolegend # 343609, CD10 APC Biolegend; #312209, CD110 PE/TexasRed BD Biosciences; #562416, CD45Ra FITC Biolegend; #304105, CD90 PE Biolegend; #328109, CD45 PerCP Biolegend; #304025, CD123 BV785 Biolegend; #306031, CD38 BV510 Biolegend; #356611, CD3 APC/Cy7 Biolegend; #317341, CD20 APC/Cy7 Biolegend; #302313, CD19 APC/Cy7 Biolegend; #363009, CD15 APC/Cy7 Biolegend; #323041) and washed with PBA. Samples were acquired using BD FACS ARIAIII (BD Biosciences) and the FACS Diva software (BD Biosciences, USA). Data was analyzed using FlowJo V10 (Tree Star, USA) and is presented as percentage of live CD45+Lin− CD34+.
Fc block
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Fc block is a laboratory equipment product designed to prevent non-specific binding of antibodies to Fc receptors. It helps to minimize background signals and improve the specificity of immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6 cytometer, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Fc block, by BD (2 mentions)
Macsquant 10, by Miltenyi Biotec (2 mentions)
Lsrfortessa analyzer, by BD (2 mentions)
Live dead, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Cd11b, by Miltenyi Biotec (2 mentions)
F4 80, by Miltenyi Biotec (2 mentions)
Cd11c, by Miltenyi Biotec (2 mentions)
Propidium iodide, by Miltenyi Biotec (2 mentions)
Deoxyribonuclease, by Worthington (2 mentions)
Mechanical tissue homogenizer, by Miltenyi Biotec (2 mentions)
Neutral buffered protease, by Worthington (2 mentions)
Collagenase t2, by Worthington (2 mentions)
Pharmlyse b, by BD (2 mentions)
Anti pe magnetic beads, by Miltenyi Biotec (2 mentions)
Aria 3, by BD (2 mentions)
Suitable species specific fluorescent secondary antibodies, by Thermo Fisher Scientific (2 mentions)
47 protocols using fc block
1
Immunophenotypic Characterization of BM-MNCs
2
Phenotypic Characterization of Immune Cells
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For phenotypic cell characterization with flow cytometry and fluorescence-activated cell sorting, cells were stained with antibodies Ly6G-PE, Ly6C-APC, CD11b-PE-Vio770, CD150(SLAM)-APC, CD48-PE, CD41-FITC, Lin-PerCP-Cy5.5, c-Kit (CD117)-PE-Vio770, B220(CD45R)-APC-Vio770, CD19-PE-Vio770 or APC-Vio770, CD43-PE, surf. IgM-FITC, surf. IgD-APC, CD21/35-FITC, CD23-APC, AA4.1(CD93)-PE-Vio770, CD5-PE, CD3-APC-Cy7, CD4-PerCP-Cy5.5 or FITC, CD8a-FITC, CD44-PE, CD62L-APC, FoxP3-PE, CD25-APC, CD19-PE-Vio770, together with e-Fluor or 7AAD viability dye (Miltenyi Biotec, Bergisch Gladbach, Germany, BD Biosciences, San Jose, CA, USA, and eBioscience, San Diego, CA, USA), and analyzed with a flow cytometer (FACSAria I; BD Biosciences, San Jose, CA, USA). The appropriate flow cytometry controls were applied throughout and included unstained controls, fluorescence minus one (FMO) controls, and the addition of Fc block (Miltenyi Biotec, Bergisch Gladbach, Germany) to prevent non-specific antibody binding. Instrument compensation and cell subset gates were set based on unstained and FMO controls, all based on viable forward-vs side-scatter gates. Doublets were excluded from the analysis. For gating strategies see Supplementary Figures S1–S6 .
3
Comprehensive PBMC and B-cell Profiling
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Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral venous blood using EasySep™ Direct Human PBMC Isolation Kit (Cat # 19654). B cells were enriched from hip bone marrow (BM) using EasySep™ Direct Human PBMC Isolation Kit (Cat # 19654) followed by EasySep Human B-Cell Enrichment Kit II without CD43 Depletion (Cat # 17963) following manufacture’s protocols. Up to 1 × 106 cells per donor were incubated with Fc block (Miltenyi) on ice for 10 min, followed by staining with a mix of 31 oligo-conjugated (barcoded) antibodies (see Table S1 for full list) for 30 min on ice in custom-made RPMI-1640 media (def-RPMI-1640; deficient in biotin, L-glutamine, phenol red, riboflavin, and sodium bicarbonate), and containing 3% newborn calf serum , followed by two washes in def-RPMI-1640/0.04% BSA. Cells were resuspended at a concentration of 1200–1500 cells/uL in custom RPMI-1640/0.04% BSA and passed through a 20-um nylon filter before loading onto a Chromium Controller (10X Genomics, Pleasanton, CA).
4
Multicolor Flow Cytometry for Immune Cell Analysis
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Thawed PBMCs were stained in Brilliant Stain Buffer (BD Biosciences) for live-dead and surface markers by using anti-human antibodies. Data were collected on BD Symphony (BD Biosciences) and analyzed by using FlowJo for Mac version 10.5 (Tree Star Inc). Primary neutrophils were isolated by using the EasySep Direct Human Neutrophil Isolation Kit (StemCell Technologies, Vancouver, British Columbia, Canada) and incubated with Fc block (Miltenyi Biotec, Bergisch Gladbach, Germany) before staining with mAbs. Flow cytometric analysis was performed with a FACS BD LSR Fortessa X20 (BD Biosciences). Analysis was performed with FlowJo software (LLC). For details on the antibodies used, refer to the Supplemental Data.
5
Fc Receptor Blocking and Intracellular CD68 Staining
6
Single-cell Flow Cytometry of Blood and Tissues
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Mouse. Single-cell suspensions of BM, spleen, and tumors were prepared, and red cells were removed using ACK lysing buffer. In other experiments, cells were culture in vitro before flow cytometry analysis was performed. All antibody incubations were performed for 15 min at 4 °C in dark and all centrifugation was done at 1500 r.p.m. at 4 °C for 5 min. Usually, up to 1 × 106 cells were incubated with Fc-block (BD Biosciences) for 10 min and surface staining was performed at 4 °C for 15 min. Cells were run on an LSRII flow cytometer (BD Biosciences) and data were analyzed by FlowJo (Tristar).
Human. Single-cell suspensions from peripheral blood were incubated with Fc-block (Miltenyi) for 10 min and surface staining was performed at 4 °C for 30 min. Cells analyzed as described above. A list of antibodies used is in Supplementary Table1 .
Human. Single-cell suspensions from peripheral blood were incubated with Fc-block (Miltenyi) for 10 min and surface staining was performed at 4 °C for 30 min. Cells analyzed as described above. A list of antibodies used is in Supplementary Table
7
Flow Cytometry Profiling of IFN Signaling
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Cells that were analysed for pSTAT and CXCL were fixed in 2% paraformaldehyde (PFA, Electron Microscopy Sciences) before flow cytometric stainings, whereas cells analysed for IFNR and HLA were fixed in 2% PFA after the last staining. Barcoded cells from each stimulus were pooled before flow cytometric stainings. For analysis of pSTAT and CXCL, cells were permeabilized with Perm Buffer III (BD Biosciences) or 0.5% saponin (Sigma-Aldrich), respectively. Stainings were performed in PBS supplemented with 2 mM EDTA and 0.5% human serum albumin (Octapharma) and FcBlock (Miltenyi Biotec) using two different 8-color panels with fluorochrome-conjugated antibodies (see below) and gating schemes as specified in S7 Fig . The IFNAR2 mAb REA124 was validated by transfection of HEK293T cells with siRNA targeting IFNAR2 ((dTdT-)GCACCATAGTGACACTGAA-dTdT), and plasmids encoding IFNAR2b or IFNAR2c (#RC201212 and #RC238664, Origene) (S8 Fig ).
Flow cytometry data were acquired on a BD FACSCanto II instrument (BD Biosciences) and analysed with FlowJo version 10.5.3. IFN-induced read-outs were determined by subtracting the geometric mean fluorescence intensity (gMFI) in unstimulated cells from stimulated cells, i.e. ΔgMFI = gMFIIFN−gMFIunstimulated.
Flow cytometry data were acquired on a BD FACSCanto II instrument (BD Biosciences) and analysed with FlowJo version 10.5.3. IFN-induced read-outs were determined by subtracting the geometric mean fluorescence intensity (gMFI) in unstimulated cells from stimulated cells, i.e. ΔgMFI = gMFIIFN−gMFIunstimulated.
8
Comprehensive Immune Cell Analysis Protocol
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Cell suspensions were incubated for 15 min at 4°C with Fc Block (BD Biosciences) and then stained for 25 min at 4°C with the following fluorochrome-conjugated antibodies: anti-CD11c-APC-Cy7 (clone: HL3, BD Biosciences) or anti-CD11c-PE (clone REA754, Miltenyi Biotec), anti-MHC-II-VioBlue (clone: M5/114.15.2, Miltenyi Biotec), anti-CD11b-PerCP-Vio700 (clone: REA592, Miltenyi Biotec), anti-EpCAM-PE (clone: caa7-9G8, Miltenyi Biotec) or anti-EpCAM-PE-Vio770 (clone caa7-9G8, Miltenyi Biotec), anti-XCR1-APC-Vio700 (clone REA707, Miltenyi Biotec), anti-PD-L2-PE (clone MIH37, Miltenyi Biotec), CD86-APC (clone PO3.3, Miltenyi Biotec). Dead cells were excluded using Zombie Aqua Fixable Viability Kit (Biolegend). For the analysis of Fc receptor expression, cells were permeabilized using an intracellular fixation & permeabilization kit (eBioscience) and incubated for 25 min at 4°C with anti-CD16(FcγRIII)/CD32(FcγRII)-PE-Vio770 (instead of Fc Block, clone: 93, Miltenyi Biotec), anti-FcϵRIα-APC (clone: MAR-1, Miltenyi Biotec), anti-CD23-APC (clone: B3B4, Miltenyi Biotec), anti-CD64-PE-Vio770 (clone: REA286, Miltenyi Biotec). Cells were acquired on a MACSquant 10 or a MACSquant 16 flow cytometer (Miltenyi Biotec) and data were analyzed using FlowJo software using the gating strategies described in Figures S2 S3
9
Immunophenotyping of IPAH PBMC Fractions
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Peripheral blood mononuclear cell fractions were generated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare) from peripheral venous blood in 26 patients with IPAH and 29 healthy control subjects. Age and sex matching with healthy control subjects was undertaken on a 1:1 basis, with three patient samples excluded because of technically low-quality specific antibody staining. Immunophenotyping was performed using flow cytometry on fresh samples. Antibody staining panels and gating strategy were designed to detect populations of myeloid, B cells, and T cells on the basis of gating strategies proposed by the Human Immune Consortium (12 (link)) and are described in the online supplement (see Tables E1 and E2 and Figure E1 in the online supplement). Peripheral blood mononuclear cell fractions were blocked with Fc block (Miltenyi Biotec) and stained with LIVE/DEAD (Thermo Fisher Scientific) before antibody staining for 20 minutes at 4°C. Stained samples were fixed in fluorescence-activated cell sorting fixative (1% formaldehyde, phosphate-buffered saline). Samples were analyzed using a BD LSRFortessa analyzer (BD Biosciences) and analyzed using FlowJo software (version 10.0.7, BD Biosciences). Additional details of the statistical analysis and quantification of immunoglobulin concentrations and IL-21 are provided in the online supplement.
10
Flow Cytometric Analysis of TGFβR Expression
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