Relative frequency of expression of specific markers were analysed using antibodies specific for αCD3, αCD8, αCD45RA, αCD27, αCD28, αCCR7 (BD Biosciences) and αCD62L (eBiosciences, USA). Where PBMCs analysed, Fc receptor block was used (Miltenyi Biotec, Germany). To detect DMF5 TCR expression, a MART-1/HLA*0201 antigen specific pentamer was used.
Fc receptor block
Manufactured by Miltenyi Biotec
Sourced in Germany
The Fc receptor block is a laboratory equipment used to prevent nonspecific binding of antibodies to Fc receptors. It works by blocking Fc receptors, which are cell surface proteins that can interact with the Fc region of antibodies, thereby reducing the potential for false-positive or background signals in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix, by BD (2 mentions)
Facscanto analyzer, by BD (2 mentions)
αcd62l, by Thermo Fisher Scientific (1 mentions)
αcd28, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Diva software, by BD (1 mentions)
Fixable viability stain 620, by BD (1 mentions)
Zombie aqua 516, by BioLegend (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Cd36 fitc, by BD (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Cd8 percp cy5, by BD (1 mentions)
Streptavidin buv395, by BD (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Pacific orange, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
16 protocols using fc receptor block
1
Multiparametric Flow Cytometry Analysis
2
Flow Cytometric Quantification of L-selectin
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All flow cytometry (FACS canto, BD Biosciences) and sorting of matched-expressing cell lines (using FACSAria II or MoFlo) were conducted within the Biomedical Research Facility at King's College London.
Cells were labelled with antibody on ice for 30 min, in solution containing Fc Receptor block (Miltenyi Biotec). All washes were performed using 10% (v/v) FCS in Hanks' balanced salt solution (lacking Mg2+ and Ca2+) with 25 mM HEPES pH 7.4. Cells were subsequently washed and labelled with either DREG56-PE (Santa Cruz Biotechnology, sc-18851) or IgG1-PE isotype control mouse IgG1 (Santa Cruz Biotechnology, sc-2866) both at 1:80 in 50 μl (∼250 ng/106 cells). Cells were washed three times before analysis of protein expression by flow cytometry. A minimum of 20,000 events were counted for each experiment. Data from flow cytometry experiments are represented as the percentage of L-selectin remaining relative to carrier-only treated cells after correction for background fluorescence with isotype-matched control antibodies. All values were normalized against IgG isotype controls.
Cells were labelled with antibody on ice for 30 min, in solution containing Fc Receptor block (Miltenyi Biotec). All washes were performed using 10% (v/v) FCS in Hanks' balanced salt solution (lacking Mg2+ and Ca2+) with 25 mM HEPES pH 7.4. Cells were subsequently washed and labelled with either DREG56-PE (Santa Cruz Biotechnology, sc-18851) or IgG1-PE isotype control mouse IgG1 (Santa Cruz Biotechnology, sc-2866) both at 1:80 in 50 μl (∼250 ng/106 cells). Cells were washed three times before analysis of protein expression by flow cytometry. A minimum of 20,000 events were counted for each experiment. Data from flow cytometry experiments are represented as the percentage of L-selectin remaining relative to carrier-only treated cells after correction for background fluorescence with isotype-matched control antibodies. All values were normalized against IgG isotype controls.
3
Neutrophil Phenotyping from Blood and BAL
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Neutrophil phenotyping was performed on isolated blood neutrophils and the BAL cell pellet (without previous neutrophil purification). Cells were treated with Fc receptor block (Miltenyi Biotec) and Fixable Viability Stain 620 (BD Biosciences) or Zombie Aqua 516 (BioLegend) for 15 minutes at room temperature. Subsequently, cells were washed with flow cytometry buffer (PBS + 2% v/v FCS + 2 mM EDTA) and stained with fluorescently labeled antibodies. Antibodies used in this study were titrated in-house and are listed in Supplemental Table 1 . Following incubation for 25 minutes (on ice), cells were washed with flow cytometry buffer and fixed with BD Cytofix (BD Biosciences). Results were analyzed using a BD LSRFortessa X-20 (BD Biosciences) equipped with Diva software (BD Biosciences). FlowJo software (BD Biosciences) was used for downstream analysis. Neutrophils were gated as CD16+CD66b+ cells within the population of living single cells (Supplemental Figure 10 ).
4
Multiplexed PBMC Barcoding and Staining
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Following stimulation, PBMCs were resuspended by pipetting and immediately fixed at RT for 10 min by adding prewarmed PFA (Electron Microscopy Sciences (Hatfield, PA, USA)) at a final concentration of 1.5%. PBMCs were then centrifuged at 1000 g for 5 min 4°C and resuspended by vortexing in 50 μL PBS before drop wise addition of 1 mL ice cold methanol and incubation on ice for 30 min. The permeabilized cells were kept overnight at −80°C. After washing with PBS, the PBMCs were stained according to a 3 × 3 barcoding grid (9 stimulation conditions) using 3 levels of pacific orange (PO) and pacific blue (PB) succinimidyl ester dyes (PB 100, 25 and 6.3 ng/mL; PO 250, 70 and 0 ng/mL; Life Technologies, Grand Island, NY, USA) for 30 min in the dark at 4°C. Barcoded PBMCs were then washed once with PBS containing 1% BSA, before being combined into one sample. The sample was washed and incubated with 2 μL Fc receptor block (Miltenyi Biotec, Bergisch Gladbach, Germany) per 1 × 106 cells for 10 min on ice. Following, the sample was subdivided into three parts and incubated for 30 min at RT in the dark with the three different antibody staining panels. An aliquot of the barcoded cells was collected before addition of antibody as a barcoding only control. The samples were then washed twice and re‐suspended in PBS containing 1% BSA and 2mM EDTA (Sigma‐Aldrich) prior to analysis.
5
Multiparametric Flow Cytometry Assay
6
Immunophenotyping of NKG2D-CART cells
7
Cryopreserved PBMC Staining Protocol
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Before staining, cryopreserved PBMCs were rapidly thawed using a water bath set to 37°C and washed once in cold PBS (without calcium and magnesium, Lonza) containing 5% AB serum and Benzonase® Nuclease (1:10,000; Merck Millipore) by centrifugation at 450 x g for 5 min at 4°C. The PBMCs were then resuspended in cold PBS and stained with pacific orange (250 ng/ml; Life Technologies) for 20 min on ice in the dark. Following live/dead staining, cells were washed once, taken up in cold FACS-buffer (PBS containing 0.5% BSA) and incubated with 2 µl Fc receptor block (Miltenyi Biotec) per 1 x 106 cells for 10 min on ice. Cells were then subdivided into two parts and incubated for 30 min on ice in the dark with the respective antibody staining panel. The samples were subsequently washed once and re-suspended in FACS-buffer prior to analysis at the flow cytometer.
8
Flow Cytometric Analysis of Intracellular Cytokines
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Following stimulation, cells were suspended in FACS staining buffer (PBS, 0.5% BSA, and 0.01% azide) and treated for 15 min with Fc receptor block (Miltenyi Biotec, San Diego, CA) and surface-labeled with PE- or APC-conjugated Abs for 20 min at 4 °C. Cells were permeabilized, stained and fixed using Cytofix/Cytoperm™(BD Biosciences San Diego, CA) according to manufacturer’s protocols. Intracellular cytokines (IFNγ or IL-4) were stained using FITC-conjugated antibodies and analyzed for expression of cytokines by flow cytometry, (CyAn flow cytometer, DakoCytomation, Fort Collins, Colorado) using Summit Acquisition Software, Version 4.2.
9
Comprehensive Flow Cytometry Characterization
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Flow cytometry analyses were performed using a FACSCanto analyzer (BD Biosciences), equipped with DIVA Software. Between 100,000 and 500,000 cells were harvested, washed with PBS or MACS buffer (PBS pH 7.2 0.5% BSA, 2 mM EDTA), treated with Fc Receptor‐Block (Miltenyi Biotec) when antibody stained, and then resuspended in the buffer used for washing. Staining was performed in MACS buffer, incubating cells with antibodies (in the proportion indicated in the table below) for 20 minutes at 4°C in the dark. For vitality staining, 7‐aminoactinomycin D (7AAD, Sigma) was used. Anti‐murine IgG beads were used for single‐staining controls (BD Biosciences). Rainbow beads (BD Biosciences) were used to calibrate the instrument detectors, for consistent MFI measurement, for analysis performed at different times.
| Antigen | Fluorochrome | Clone | Company | Dilution |
|---|---|---|---|---|
| CD33 | BV421 | WM53 | BD Biosciences | 1:20 |
| CD235a | APC | REA175 | Miltenyi Biotec | 1:20 |
| CD34 | VioBlue | AC136 | Miltenyi Biotec | 1:25 |
| CD3 | PE‐Cy7 | HIT3a | BioLegend | 1:50 |
| CD4 | Pacific Blue | RPA‐T4 | BioLegend | 1:50 |
| CD8 | APC‐Cy7 | SK1 | BD Biosciences | 1:33 |
| CD35 | PE | E11 | BD Biosciences | 1:50 |
| CD46 | PE | 8E2 | eBioscience | 1:20 |
| CD55 | PE | JS11 | BioLegend | 1:33 |
| CD59 | PE | p282 (H19) | BioLegend | 1:33 |
| B2M | PE | 2M2 | BioLegend | 1:20 |
| MHC‐I | APC | W6/32 | Santa Cruz Biotech | 1:10 |
10
Multiparameter Immune Profiling of Tumor Allografts
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A total of 3 million cells from tumor allografts were resuspended in Maxpar Cell Staining Buffer (Fluidigm), incubated with Fc-receptor block (Miltenyi Biotec), and stained with 24 metal-conjugated antibodies. Maxpar antibodies CD45-89Y, Gr1-141, CD11c-142, CD69-143Nd, CD115-144Nd, CD4-145, F4-80-146, CD11b-148, CD19-149, CD25-151, CD3-152-Sm, CD14-156Gd, PD1-159Tb, CD40-161Dy, Tim3-162Dy, CD49b-164Dy, CD8a-168, CD206-169Tm, Nk1.1–170, CD80-171Yb, CD86-172Yb, and IA/IE-174Yb were used to detect and cluster immune cells (Fluidigm). Tumor-targeting antibodies D3-GPC2-IgG (GPC2) and dinutuximab (GD2) were conjugated with 153Eu and 154Sm using metal conjugation kits (Abcam). Cell-ID Intercalator-Ir (125 nM) was used to distinguish live versus dead cells and events were acquired using CyTOF2 mass cytometer (Fluidigm). Results were analyzed using FlowJo software.
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