Freshly isolated B cells were treated with Human Fc Receptor Binding Inhibitor (eBioscience, San Diego, CA) to block the Fc receptor prior to staining. Cells were then stained with FITC-conjugated anti-CD27, PE-conjugated anti-CD19, PE-Cy7-conjugated anti-CD20, BV421-conjugated anti-CD38 and APC-conjugated anti-CD3 (Biolegend, San Diego, CA). The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Life Technologies, Carlsbad, CA) was used to determine the viability of cells. Single plasmablasts (CD3−CD19+CD20lo/−CD27hiCD38hi) were sorted into 96-well plates with 4 μl of cell lysis buffer containing 10 mM DTT (Invitrogen, Carlsbad, CA) and RNase Inhibitor (Eppendorf) in each well (13 (link)). Cell sorting was performed using a BD InfluxTM cell sorter.
Human fc receptor binding inhibitor
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Human Fc receptor binding inhibitor is a laboratory reagent designed to block the binding of the Fc region of human immunoglobulins to their cognate Fc receptors. This product can be used in various immunological and cell-based assays to study Fc receptor-mediated functions.
Automatically generated - may contain errors
Lab products found in correlation
Cd235 apc, by BioLegend (5 mentions)
Dxp10 flow cytometer, by Cytek (5 mentions)
Cd11b pe cy5, by BioLegend (5 mentions)
Ficoll paque plus, by GE Healthcare (4 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Cellquest software, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Cd41 fitc, by BioLegend (3 mentions)
Cd71 apc, by BD (3 mentions)
Cd34 fluor450, by Thermo Fisher Scientific (3 mentions)
Facsdiva software, by BD (3 mentions)
Anti cd8a apc cy7 hit8a, by BioLegend (2 mentions)
Cd34 pe cy7, by BioLegend (2 mentions)
Gata1 pe, by Cell Signaling Technology (2 mentions)
Aria 1, by BD (2 mentions)
Attune cytometer, by Thermo Fisher Scientific (2 mentions)
Okt 9, by Thermo Fisher Scientific (2 mentions)
Attune cytometer software, by Thermo Fisher Scientific (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
36 protocols using human fc receptor binding inhibitor
1
Isolation and Characterization of Plasmablasts
2
Quantifying Myeloma Cell EPOR Levels
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EPOR on the surface of myeloma cells was stained with allophycocyanin (APC)-labelled anti-EPO receptor antibody [clone 12 K90] from Life Span Biosciences Inc (LS-C182845), 20 μl/ml for 30 min on ice after 20 min preincubation with human Fc receptor binding inhibitor (eBioscience, 50 μl/ml). Isotype control mouse IgG1 APC MG105 (Invitrogen) was used. All incubations were performed in PBS/1 % BSA. Flow cytometry was performed using LSR II (Becton Dickinson) with FACS Diva software (Becton Dickinson), and analysed with Cytobank [31 ]. Gates were set on live cells with forward and side scatter and duplets were excluded.
3
Flow Cytometric Analysis of CD235 and CD11b
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Cells were incubated with human Fc receptor–binding inhibitor (#14-9161-73; eBioscience) followed by primary antibodies CD235-APC (#306607; BioLegend) and CD11b-PE/Cy5 (#101209; BioLegend). Data were collected on a DxP10 flow cytometer (Cytek) and analyzed by using FlowJo Software, version 9.7.2 (FlowJo, LLC).
4
Immune Cell Phenotyping and Cytokine Analysis
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Before antibody staining, cells were stained with Fixable Viability Dye eFluor™ 780 (eBioscience) on ice for 30 min to exclude dead cells, followed by treatment with a human Fc receptor-binding inhibitor (eBioscience) for 15 min at room temperature. Surface proteins were stained on ice for 30 min with the following fluorochrome-conjugated antibodies: antihuman CD3 (clone SK7, eBioscience), anti-human CD4 (clone RPA-T4, BioLegend), and antihuman CD8 (clone RPA-T8, eBioscience). For intracellular staining, cells were fixed and permeabilised with a FoxP3 staining buffer kit (Thermo Fisher Scientific) and stained with the following fluorochrome-conjugated antibodies: antihuman interferon-γ (IFN-γ) (clone B27, BD Biosciences), and antihuman tumour necrosis factor-α (TNF-α) (clone Mab11, BD Biosciences). Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter), and the results were analysed using the FlowJo software (Tree Star Inc.).
5
Multi-Marker Flow Cytometry Analysis
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The following antibodies and reagents were used for sample analysis by flow cytometry: Human Fc receptor binding inhibitor (eBioscience), Anti-CD45 PE-Cy7 (HI30; eBioscience), anti-CD11b PE (M1/70; BD Biosciences), anti-CD56 BV421 (HCD56; Biolegend), anti-CD3 PerCp-Cy5.5 (OKT3, eBioscience), anti-CD4 Alexa Fluor 700 (OKT4; eBioscience), anti-CD8a APC-Cy7 (HIT8a; Biolegend), anti-Vα24 PE (C15; Beckman Coulter), anti-Vβ11 FITC (C21; Beckman Coulter), anti-BDCA-2 APC (201A; Biolegend), anti-TCRγδ PE-CF594 (B1; BD Biosciences), anti-CD11c BV421 (3.9; Biolegend), anti-HLA-DR PE-CF594 (G46-6; BD Biosciences), anti-Cytokeratin 10 biotin (DE-K10; Abcam), anti-Cytokeratin 14 FITC (LL002; Abcam), Streptavidin-APC-Cy7 (BD Biosciences), and the anti-Human FoxP3 Staining Set (236A/E7; eBioscience). Rat IgG2a,κ Isotype Control APC (R35-95; BD Biosciences) was used as an isotype control for intracellular FoxP3 staining. Dead cells were excluded using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, New York, U.S.) according to the manufacturers’ instructions, and anti-CD45 was used to differential immune cells from non-immune cells.
6
Phenotypic Profiling of Immune Cells
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Fresh mucosa samples were minced and filtered through a sterile Nylon Filter (BD Falcon). The single cell suspension was incubated with human Fc Receptor binding inhibitor (eBioscience) for 20 min, pelleted, and stained in FACS buffer (phosphate‐buffered saline/2% Flow cytometry staining/0.02% sodium azide) for 30 min at 4°C. The following fluorochrome‐conjugated antibodies were used: anti‐human CD8A PE (clone HIT8a), anti‐human CD28 FITC (clone CD28.2), anti‐human CD80 FITC (clone 2D10.4), anti‐human HLA ABC FITC (clone W6/32) all from eBioscience and anti‐pan Cytokeratin PE (clone C‐11; Abcam). Stained samples were acquired on a FACSCalibur based on CellQuest software (Becton Dickinson). The percentage of cells positive for the molecules of interest was reported.
7
Cell Surface Flow Cytometry Analysis
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For cell surface flow cytometry, cells were incubated with human Fc receptor binding inhibitor (No. 14-9161-73, eBioscience, San Diego, CA) followed by primary antibodies CD235-APC (No. 306607, BioLegend, San Diego, CA), CD41-FITC (RRID: AB_314373, BioLegend No. 303703), and CD11b-PE/Cy5 (No. 101209, RRID: AB_312995, BioLegend) or CD-71-APC (No. BD551374, RRID: AB_398500, BD Biosciences, Franklin Lakes, NJ), CD34-Fluor450 (No. 48-0341-82, RRID: AB_468936, eBioscience), CD34-PE/Cy7 (No. 343615, RRID: AB_2629725, BioLegend), and GATA1-PE (No. 13353, RRID: AB_2798187, Cell Signaling Technology, Danvers, MA). Data were collected on a DxP10 flow cytometer (Cytek, Cerritos, CA) and analyzed using FlowJo Software, Version 9.7.2.
8
Phenotypic Analysis of Dendritic Cells
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Cells were collected in phosphate buffered saline (PBS) and pre-incubated with Fc receptor blocking anti-mouse CD16/CD32 (clone 93) or human Fc receptor binding inhibitor (eBioscience Inc., USA). A blue fluorescent amine-reactive dye (Invitrogen, USA) was used as live/dead cell marker. Murine cells were decorated with anti-mouse CD80 (clone 16-10A1, APC-conjugated), CD86 (clone GL1, PE-conjugated), I-Ab (clone AF6-120.1, FITC-conjugated), CD11c (clone N418, PE-Cy7-conjugated); human cells with anti-human CD40 (clone 5C3, PE-conjugated), CD54 (clone HA58, APC-conjugated), CD80 (clone 2D10, APC-conjugated), CD83 (clone HB15e, PE-conjugated), CD86 (clone IT2.2, PE-Cy7-conjugated), CD11c (clone 3.9, Brilliant Violet 711-conjugated) (BioLegend, USA). Flow cytometry analysis was performed using an LSR-II and the software FACSDiva (BD Bioscience, USA) and FlowJo Mac v9.6 (Tree Star, Inc., USA). Forward and sideward scatter gating was used to restrict fluorescence analysis to intact single cells only. Live cells were gated based on their lower blue fluorescence emission of the live/dead cell marker and DCs were identified by their enhanced surface expression of CD11c. Activated cells were identified by gating for cell populations with enhanced fluorescence intensity indicative of the antibody-detected surface molecules.
9
Isolation and Sorting of Mononuclear Cells
10
Immune Cell Profiling of Tumor Samples
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Tumor and corresponding normal mucosa tissue samples were mechanically dissected and passed through a sterile nylon filter (BD Falcon, Heidelberg, Germany). The single cell suspension was pelleted, suspended in FACS buffer (PBS/2% FCS/0.02% sodium azide) and stained with fluorochrome‐conjugated Abs (Table S1 ). Staining was undertaken in flow cytometry buffer for 30 minutes at 4°C after 20 minutes of incubation with human Fc receptor binding inhibitor (eBioscience, San Diego, CA, USA). After two washes, samples results were acquired on a FACSCalibur based on CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). At least 10 000 cells were analyzed for each sample.
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