Multi-color flow cytometry analysis was performed on PBMCs from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, ICOS-PE, HLA-DR-PerCP-Cy5.5, CD25-PE-Cy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, PD-1-PE, Tim-3-AF700, CD4-APC-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7 (R&D Systems, Minneapolis, MN), CTLA-4-FITC (LSBio, Seattle, WA) and FoxP3-APC (eBioscience, San Diego, CA) for T cells. For natural killer (NK) cells, CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 (BD) were used. For myeloid-derived suppressor cells (MDSCs), CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD) were used. 1×105 cells were acquired on an LSRII (BD), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.
Cd3 v450
Manufactured by BD
Sourced in United States
The CD3-V450 is a fluorochrome-conjugated antibody used in flow cytometry applications. It is designed to detect and quantify CD3-positive cells, which are a key component of the T cell lineage. The CD3-V450 antibody is conjugated to the V450 fluorescent dye, providing a specific signal for the identification and analysis of CD3-expressing cells.
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Lab products found in correlation
Cd8 fitc, by BD (5 mentions)
Cd8 apc h7, by BD (5 mentions)
Facscanto 2, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Hla dr v500, by BD (3 mentions)
Cd45ra pe cy7, by BD (3 mentions)
Cd127 pe, by BD (2 mentions)
Ccr7 pe cy7, by BD (2 mentions)
Foxp3 apc, by Thermo Fisher Scientific (2 mentions)
Cd38 pe, by BD (2 mentions)
Iga fitc, by Bio-Rad (2 mentions)
Igm fitc, by Bio-Rad (2 mentions)
Igm apc, by BD (2 mentions)
Cd20 percp cy5, by BD (2 mentions)
Cd38 pe cy7, by BD (2 mentions)
Cd19 apc, by BD (2 mentions)
Cd19 brilliant violet 421, by BioLegend (2 mentions)
Cd27 apc h7, by BD (2 mentions)
Dntps, by New England Biolabs (2 mentions)
Ribolock, by Thermo Fisher Scientific (2 mentions)
26 protocols using cd3 v450
1
Multiparametric Flow Cytometry Analysis
2
Tolerogenic Dendritic Cell Modulation of T-Cell Subsets
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After 5 days of culture, PBMC co-cultured with mature DC (mDC), VitD3-tolDC (100% VitD3-tolDC) and 50% VitD3-tolDC (50% VitD3-tolDC + 50% mDC) in the presence and absence of IFN-beta were harvested and stained using CD3-V450, CD4-PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Bioscience), CD183 AF488, CD196 BV605 and CD45 AF700 (Biolegend, San Diego, CA, USA) for T cell analysis; and CD4 PerCP-Cy5.5, CD25 PE, CCR4 PE-Cy7, CD127 AF647, CD45RO APC-H7, CD3 V450, HLA-DR V500 (BD Biosciences) and CD45 AF700 (Biolegend) for Treg analysis. Monoclonal antibodies were incubated for 20 min at room temperature and protected from the light. Samples were washed and a total of 50,000 CD3+ events were acquired on an LSR Fortessa flow cytometer (BD Biosciences). Both panels were analyzed using FACSDiva software (BD Biosciences). The gating strategy used to analyze the desired T cell subpopulations was previously reported [28 (link)].
3
Analysis of Tumor-Specific T-Cell Responses
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B16-Ova cells (106) solved in 200 μl Ringer's solution were injected into the right shaved flank of female OT1 mice at day 0. The tumors were treated as described above for the other mice. At day 14, the draining lymph nodes (axillary and inguinal) were removed and single cell suspensions were prepared with cell strainers (pore size of 70 μm). For detection of DCs present in the lymph nodes and presentation of the model tumor antigen, the cells were stained with CD11c-FITC (BD Pharmingen), CD8-APC (BD Pharmingen), OVA257-264 (SIINFEKL)-PE (eBioscience) and CD3-V450 (BD Pharmingen). T-cell activation was measured by analyzing intracellular IFNγ after re-stimulation of the cells. For this, 2 × 106 cells were re-stimulated with OVA peptide (10-7M) and Golgi Plug for 5 h. Surface staining was performed with CD3-V450 (BD Pharmingen) and CD8-PE (BD Pharmingen) by incubating for 30 min at 4 °C. After washing, the cells were made permeable by addition of Cytofix/Cytoperm and incubation for 20 min at 4 °C. For intracellular staining, the antibody IFNγ-Pe-Cy7 (BD Pharmingen) was added and the cells were incubated for another 30 min at 4 °C. The cells were washed and analyzed by multicolor flow cytometry with the Gallios Flow Cytometer (Beckman Coulter Inc.).
4
Isolation and Identification of T-cell Subsets
5
Comprehensive Immune Phenotyping of PBMC
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Immune activation and differentiation were quantified as previously described [54] (link). In brief, one million thawed PBMC were stained with either an activation or differentiation panel for 15 minutes at 37°C prior to fixation in formaldehyde. Both panels included CD3 V450 (Becton Dickinson); CD4 PE-Texas Red (Invitrogen); CD8 Qdot605 (Invitrogen). Activation panel included HLA-DR FITC; PD-1 AF647; CD38 PE; CCR5 PE-Cy5; 45RA PE-Cy7 (all Becton Dickinson); CCR7 APC eFluor-780 (eBioscience). Differentiation panel included CD45RA PE; CD28 PE-Cy5; CCR7 PE-Cy7; CD31 FITC (all Becton Dickinson); CD57 AF647 (Biolegend); CD27 AF780 (eBioscience). For Tregs, PBMC were surface stained with CD4 PerCP, CD127 PE and CD25 FITC (all Becton Dickinson) followed by intracellular staining using eBioscience FoxP3 staining kit and FoxP3 APC as per manufacturer's instructions. Data was acquired on a BD LSR-Fortessa and analysed using FlowJo version 10.
6
Isolation of Influenza-specific Plasmablasts
7
Flow Cytometric Analysis of T Cell Subsets
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Blood samples were collected prior to the first administration of the treatment (baseline) and processed by a centralized laboratory within the first 24 h after their collection. Samples of whole blood were analyzed by flow cytometry to determine the percentage and absolute number of T lymphocyte subpopulations using the following combination of monoclonal antibodies per panel: CD3-V450, CD4 PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Biosciences), CD183 AF488, CD196 BV605, and CD45 AF700 (BioLegend, San Diego, CA, USA). The absolute cell number quantification was performed as previously reported [16 (link)]. Samples were acquired on a LSR II Fortessa flow cytometer (BD Biosciences, San José, CA, USA).
The following T cell subpopulations were analyzed: CD4+ naïve, CD4+ TCM, Th1CM, Th1Th17CM, Th2CM, Th17CM, CD4+ TEMRA, CD4+ TEM, Th1EM, Th1Th17EM, Th2EM, Th17EM, CD8+ naïve, CD8+ TCM, CD8+ TEMRA, CD8+ TEM, double positive (CD4+CD8+), and double negative (CD4−CD8−) T cells. Analysis was performed using the FACSDiva software (BD Biosciences). The gating strategy for the subpopulations analyzed in whole blood is shown inFigure 1 and previously described by Quirant-Sánchez et al. [22 (link)].
The following T cell subpopulations were analyzed: CD4+ naïve, CD4+ TCM, Th1CM, Th1Th17CM, Th2CM, Th17CM, CD4+ TEMRA, CD4+ TEM, Th1EM, Th1Th17EM, Th2EM, Th17EM, CD8+ naïve, CD8+ TCM, CD8+ TEMRA, CD8+ TEM, double positive (CD4+CD8+), and double negative (CD4−CD8−) T cells. Analysis was performed using the FACSDiva software (BD Biosciences). The gating strategy for the subpopulations analyzed in whole blood is shown in
8
T Cell Inhibitory Receptor Profiling in RA
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Peripheral blood mononuclear cells from HCs and paired PBMCs and SFMCs from RA patients were stained for the presence of T cell co-inhibitory receptors. Non-specific binding was blocked by 50 µg/ml mouse IgG (Jackson), and surface staining was performed using the following antibodies: CD3 V450 (clone: UCHT1, BD), CD4 PE-CF594 (clone: RPA-T4, BD), CD8 BV785 (clone: RPA-T8, BioLegend), CD25 Alexa 700 (clone: BC96, BioLegend), Tim-3 BV711 (clone: F38-2E2, BioLegend), CTLA-4 PerCPCy5.5 (clone: L3D10, BioLegend), Tigit PE-Cy7 (clone: MBSA43, eBioscience), PD-1 APC (clone: MIH4, BD), Live-dead near IR (Thermo Fisher Scientific). Cells were permeabilized by BD Facs lysing solution and BD Facs Perm Solution 2 (both BD bioscience). Intracellular staining was performed using Eomes PE (clone: WD1928 ebioscience). All antibodies were used in the concentration recommended by the manufacturer. Gating was done on lymphocytes, excluding doublets and dead cells. Gates were set using FMOs. CD3+ CD4+ and CD3+ CD8+ cells were investigated for their expression of T cell co-inhibitory receptors.
9
Blocking Cytokines and Checkpoint Inhibitors in T-cell Assays
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Cells were thawed, washed and reconstituted in AIM V serum-free medium (Life Technologies, Oslo, Norway) containing 0.1% human serum albumin. After an overnight rest, cells were pulse-labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Life Technologies) at a concentration of 2 μM for 5 minutes. The following blocking antibodies were added to parallel culture wells at a final concentration of 10 μg/mL: anti-IL-10 (R&D Systems, MN, USA; clone 23738), anti-TGF-β (R&D; clone 1D11), anti-PD-L1 (eBioscience, CA, USA; clone MIH1), and anti-HVEM (R&D; clone 94801).
After a 30 minute incubation, cultures were stimulated with either Gag or Env 15-mer overlapping peptide panels (NIH AIDS Research and Reference Reagent Program, MD, USA) at a final concentration of 2 μg/mL/peptide. Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich, MO, USA) at a final concentration of 0.5 μg/mL was used as a positive control.
Cells were cultured at 37°C in 5% CO2 for 5 days, harvested, and stained with the following fluorochrome-conjugated antibodies: CD3 V450, CD8 APC-H7, HLA-DR BV605, CD45RA APC (all BD) and CD25 PE (Biolegend). 7-aminoactinomycin D (7-AAD, BD) was added for dead cell exclusion. Flow cytometry data were acquired on a BD FACS Canto II with BD Diva 6.1 software, and analyzed in FlowJo X (FlowJo LLC, OR, USA).
After a 30 minute incubation, cultures were stimulated with either Gag or Env 15-mer overlapping peptide panels (NIH AIDS Research and Reference Reagent Program, MD, USA) at a final concentration of 2 μg/mL/peptide. Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich, MO, USA) at a final concentration of 0.5 μg/mL was used as a positive control.
Cells were cultured at 37°C in 5% CO2 for 5 days, harvested, and stained with the following fluorochrome-conjugated antibodies: CD3 V450, CD8 APC-H7, HLA-DR BV605, CD45RA APC (all BD) and CD25 PE (Biolegend). 7-aminoactinomycin D (7-AAD, BD) was added for dead cell exclusion. Flow cytometry data were acquired on a BD FACS Canto II with BD Diva 6.1 software, and analyzed in FlowJo X (FlowJo LLC, OR, USA).
10
Leukocyte Profiling from Human Blood
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Human blood samples (0.5–3mL) were transferred into 0.1 M EDTA coated 50 mL lab tubes. Leucocytes fraction was obtained after incubating the blood samples for 2.5 minutes in room temperature with BD FACS Lysing Solution (BD bioscience, San Jose, California). Leucocytes were stained with FACS buffer containing the following conjugated antibodies (10 minutes on ice in the dark): CD3-V450, CD19-V450, CD56-V450, HLA-DR1-APC, CD14-APC-Cy7 and CD16-FITC (BD Bioscience). Stained samples were further washed and filtered using 70μm mesh before analyzed by FACS (FACS Canto II; BD Biosciences, San Jose, California) and the FlowJo software.
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