A total of 106 PBMCs were plated in 96-well round-bottom plates in RPMI + 10% FBS (Gemini) + 1% penicillin, streptomycin, and l -glutamine (Invitrogen, Carlsbad, CA). After centrifugation and removal of media, cells were surface stained with 50 μL of a cocktail mix consisting of titrated volumes of LIVE/DEAD violet dye (Life Technologies, Grand Island, NY); anti-CD3, anti-CD14, and CD16 Pacific Blue; anti-CD19 PcP-Cy5.5; anti-CD27 APC; anti-IgD PE; anti-CD20 FITC; anti-CD24 PE-Cy7 (BD Biosciences, San Jose, CA); and anti-CD38 APC-AF700 (Beckman Coulter, Brea, CA) (table 2 ). A “dump” channel consisting of LIVE/DEAD dye and CD3, CD14, and CD16 Pacific Blue was used to exclude dead cells, T cells, and monocytes. After fixing the cells with 1% paraformaldehyde (PFA), they were acquired on a LSRII flow cytometer (BD Biosciences, San Jose, CA).
Anti cd27 apc
Manufactured by BD
Sourced in United States
Anti-CD27-APC is a fluorescently labeled monoclonal antibody that binds to the CD27 antigen. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on various immune cells. The APC (Allophycocyanin) fluorescent label allows for detection and analysis of CD27-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45 v500, by BD (3 mentions)
Anti igd pe, by BD (2 mentions)
Anti cd20 fitc, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd21 pe, by BD (2 mentions)
Anti cd19 pe cy7, by BD (2 mentions)
Facs lysing solution, by BD (2 mentions)
Anti igd v450, by BD (2 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Live dead violet dye, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by BD (1 mentions)
Anti cd19 pcp cy5, by BD (1 mentions)
Anti cd24 pe cy7, by BD (1 mentions)
Indo 1 am, by Thermo Fisher Scientific (1 mentions)
Anti cd19 pe cy7, by BioLegend (1 mentions)
Anti cd45ro pe, by BD (1 mentions)
Anti cd4 percp cy5, by BioLegend (1 mentions)
11 protocols using anti cd27 apc
1
Multi-parameter Immune Cell Profiling
2
Calcium Flux Profiling of T and B Cells
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Previously frozen PBMCs were incubated with the cell-permeant dye indo-1, AM (Molecular Probes) for 45 min at 37°C and then surface-stained with anti–CD8 PE/Cy5 (BioLegend), anti–CD27-APC (BD Biosciences), anti–CD45RO-PE (BD Biosciences), anti–CD45RA-FITC (BioLegend), anti–CD4-PerCP/Cy5.5 (BioLegend), and anti–CD19-PE/Cy7 (BioLegend). The indo-1 fluorescence ratio was acquired as a function of time on an LSR II flow cytometer, and kinetics curves were generated using the FlowJo software. Collection of a 30-s or 1-min baseline was followed by stimulation with 10 μg/mL soluble anti-CD3 (clone UCHT1, BioLegend), 20 μg/mL anti-κ + anti-λ F(ab’)2 (SouthernBiotech), or 1 μg/mL ionomycin. All the calcium flux profiles were generated using a standard protocol.
3
Phenotypic Characterization of T-Cell Subsets
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque (GE Healthcare, Sweden) and analyzed by flow cytometry. PBMCs were stained with fluorochrome-conjugated monoclonal antibodies against surface antigens for 30 min at 4 °C. The antibodies used included anti-CD3-PE-Cy7, anti-CD4-Horizon V500, anti-CD8-APC-Cy7, anti-CD31-FITC, anti-CXCR4-PerCP-Cy5.5, and anti-CD28-APC-H7 (all from BD Biosciences, San Jose, California, USA). Phenotypic characterization of CD28null-Tang and CD28+-Tang subsets was performed by extracellular staining with anti-CD57-APC, anti-CD27-APC, anti-CCR7-APC, or antihTERT-APC or the corresponding isotype controls (all from BD Biosciences). Multicolor flow cytometry was performed using an CytoFlex S (Beckman Coulter), and FlowJo V10 software (Treestar, San Carlos, California, USA) was used to analyze the data.
4
Phenotypic Characterization of B-Cell Subsets
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A total of 0.5 x 106 PBMCs were stained with the following combination of antibodies: Anti-CD19-BV421 (clone HIB19), anti-CD20-PerCPCy™5.5 (clone L27), anti-CD21-PE (clone B-ly4), anti-CD27-APC (clone M-T271) and anti-CD45-V500 (clone HI30). Anti-CD19-BV421, anti-CD20-PerCPCy™5.5, anti-CD21-PE, anti-CD27-APC, anti-CD45-V500, PerCP-Cy5.5 mouse IgG1 k isotype control, BV421 mouse IgG1 k isotype control, V500 mouse IgG1 k isotype control, PE mouse IgG1 k isotype control, and APC mouse IgG1 k isotype control were purchased from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, USA). B-cell populations were identified based on the expression of the following markers: activated memory B-cells (CD19+ CD20+ CD21- CD27+ CD45+), resting memory B-cells (CD19+ CD20+ CD21+ CD27+ CD45+), tissue-like memory cells (CD19+, CD20+ CD21- CD27- CD45+), and resting naïve B-cells (CD19+ CD20+ CD21+ CD27- CD45+).
CD34+ hematopoietic progenitors in peripheral blood were enumerated using the Stem Cell Enumeration Kit (Becton Dickinson).
All flow cytometry analyses were performed using a BD LSRFortessa™ Flow Cytometer (Becton Dickinson) from the I2MC cytometry platform (CHU Rangueil, Toulouse, France) and BD FACSDiva™ Software (Becton Dickinson).
CD34+ hematopoietic progenitors in peripheral blood were enumerated using the Stem Cell Enumeration Kit (Becton Dickinson).
All flow cytometry analyses were performed using a BD LSRFortessa™ Flow Cytometer (Becton Dickinson) from the I2MC cytometry platform (CHU Rangueil, Toulouse, France) and BD FACSDiva™ Software (Becton Dickinson).
5
Phenotyping of B-cell Subsets in PBMC
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B-cell subsets were measured in freshly thawed cryopreserved PBMCs. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD19-PE-Cy5, anti-CD21-PE, anti-CD20-PE-Cy7, anti-CD27-APC, anti-IgM-FITC, anti-CD38-FITC, anti-HLA-DR-PE-Cy7 (BD Bioscience); anti-CD10-APC-Cy7, anti-BAFFr-APC-Cy7 (Biolegend); anti-TACI-PE, anti-CxCr5-APC (R & D Systems) and analyzed with Guava easyCyte 8HT (Millipore) and FlowJo (Treestar). Subsets were expressed as percentages of the parent CD19+ B-cell population.
6
Isolation and Sorting of Human B Cell Subsets
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About 50 mL of healthy human donor one (HD1) or healthy donor two (HD2) source leukocytes peripheral blood (Gulf Coast Regional Blood Center, Houston, TX, USA; E5318 product code) was used as a source for separating all nucleated cells from red blood cells using HetaSep (Stemcell Technologies) according to the manufacturer’s protocol. B cells were then extracted and enriched for from the nucleated cells using an EasySep Human CD19 + Positive Selection Kit (Stemcell Technologies) according to the manufacturer’s protocol. The enriched B cells were resuspended in ~500 µL of FACS buffer. These B cells were then stained with a 1:16 dilution of anti-IgD-PE (BD, cat#562024), a 1:16 dilution of anti-CD20-FITC (BD, cat#560962), a ~1:26 dilution of anti-CD19-v450 (BD, cat#560353), a 1:61 dilution of anti-CD3-PerCP-Cy5.5 (Biolegend, cat#300327), and a 1:16 dilution of anti-CD27−APC (BD, cat#558664) and then sorted for naive (CD20+CD19+IgD+CD3−CD27−) and memory (CD20+CD19+IgD−CD3−CD27+) cells on a FACSAria II.
7
Plasmablasts Enrichment and Characterization
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Blood was collected into tubes containing heparin. To assess plasmablasts frequency in PBMCs for analytical flow cytometric studies, PBMCs were enriched from whole blood (day 10 after first, and day 7 after second vaccination) using direct PBMCs isolation kit (StemCell Technologies). For singe-cell antibody secretion and paired antibody sequencing studies, plasmablasts were enriched from the whole blood (day 7 after second vaccination) by negative selection using custom direct human plasmablasts isolation kit containing paramagnetic beads and antibodies for negative selection (StemCell Technologies). Enriched cells were stained 30 min on ice in a RoboSep buffer (StemCell Technologies) containing following phenotyping antibodies; anti-CD19-FITC (1:20 dilution, eBioscience), anti-CD27-APC (1:20 dilution), and anti-CD38-PE (1:25 dilution, BD Biosciences), and then analyzed by flow cytometry using an SH800 cell sorter (Sony). A DAPI stain was used as a viability dye to exclude dead cells. Plasmablasts were identified as DAPI-CD19loCD27hiCD38hi cells. Approximately 40,000 and ∼6,000 plasmablasts were FACS-sorted in a bulk for paired antibody sequencing and single-cell antibody secretion studies, respectively.
8
Monoclonal Antibody Characterization of MoDC and γδ T Cells
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The following monoclonal antibodies were used to characterize MoDC: anti-CD86 FITC, anti-CD1a PE, anti-HLA-DR PERCP, anti-CD83 APC, anti-CD14 APC-H7, anti-CD80 FITC, anti-CD40 PE, anti-HLA-I APC, anti-CCR7 PE-Cy7, anti-CCR5 APC-H7 from BD Biosciences, and anti-BT3A.1 PE from BioLegend. To evaluate γδ T lymphocytes phenotype we used anti-Vδ2 FITC, anti-CD3 PE, anti-CD69 PERCP, anti-CD45RA PE-Cy7, anti-CD27 APC, anti-CD16 PACIFIC BLU, anti-CD25 APC-H7 (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software (TreStar, Olten, Switxìzerland).
9
Multiparametric Flow Cytometry Analysis
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Cell surface marker expression was analyzed by flow cytometry using a BD FACSLyric cytometer and data analysis was performed with the FlowJo software, both from Becton-Dickinson.
A surface staining protocol was applied to study the distribution of different cell subpopulations in peripheral blood. Briefly, 50 μL of peripheral whole blood were incubated with different combinations of fluorochrome conjugated monoclonal antibodies 20 min at room temperature (25°C). Red blood cells were lysed for 10 min with 2 mL of FACS Lysing solution (Becton Dickinson) and washed with phosphate-buffered saline (PBS) before flow cytometry analysis. Combinations of the following monoclonal antibodies were used: anti-CD3-PerCPCy5.5, anti-CD4-V500, anti-CD8-APCR700, anti-CD19-PECy7, anti-CD56-FITC, anti-CD45-APCH7, anti-CD45-V500, anti-HLA-DR-V450, anti-CD25-PECy7, anti-CD127-APC, anti-CD45RA-BV605, anti-CXCR5-BB515, anti-CXCR3-APC, anti-CCR6-PE, anti-CCR7-APCR700, anti-CD27-APC, anti-IgD-V450, anti-CD38-PE, anti-CD38-PerCPCy5.5, and anti-CD24-PE, all from Becton-Dickinson.
A surface staining protocol was applied to study the distribution of different cell subpopulations in peripheral blood. Briefly, 50 μL of peripheral whole blood were incubated with different combinations of fluorochrome conjugated monoclonal antibodies 20 min at room temperature (25°C). Red blood cells were lysed for 10 min with 2 mL of FACS Lysing solution (Becton Dickinson) and washed with phosphate-buffered saline (PBS) before flow cytometry analysis. Combinations of the following monoclonal antibodies were used: anti-CD3-PerCPCy5.5, anti-CD4-V500, anti-CD8-APCR700, anti-CD19-PECy7, anti-CD56-FITC, anti-CD45-APCH7, anti-CD45-V500, anti-HLA-DR-V450, anti-CD25-PECy7, anti-CD127-APC, anti-CD45RA-BV605, anti-CXCR5-BB515, anti-CXCR3-APC, anti-CCR6-PE, anti-CCR7-APCR700, anti-CD27-APC, anti-IgD-V450, anti-CD38-PE, anti-CD38-PerCPCy5.5, and anti-CD24-PE, all from Becton-Dickinson.
10
Immunophenotyping of B-cell Subsets in CVID
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A surface staining protocol was applied to evaluate B-cell subpopulations and phenotypically classify CVID patients. Briefly, 50 µL of peripheral whole blood was incubated with a combination of fluorochrome-conjugated monoclonal antibodies (5 µL of each antibody) for 20 min at room temperature (25°C). Red blood cells were lysed for 10 min with 2 mL of FACS Lysing solution (Becton Dickinson) and washed with phosphate-buffered saline (PBS) before flow cytometry analysis. The combination of the following monoclonal antibodies was used to determine the distribution of B-cell subpopulations—anti-CD19-PECy7, anti-CD45-V500, anti-CD27-APC, anti-IgD-V450, and anti-CD21-BV605—all from Becton Dickinson.
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