Rats were intubated using an 18-gauge blunted cannula inserted below the cricoid cartilage before 1.5 ml of chilled lavage buffer (1X PBS, 0.5% BSA, 0.5 mM EDTA, 25 mM HEPES) was squirted and retrieved. This procedure was repeated for a total of 2 independent lavages. Cells were stained for viability with Live/Dead Stain (Life Technologies, Carlsbad, CA), and then stained using FITC anti-CD45. Next, cells were stained with the following cocktail of secondary conjugated anti-rat monoclonal antibodies diluted per manufacturers’ instructions: V450 anti-CD4, APC anti-CD3, PE-Cy5 anti-CD45RA, PE-Cy7 anti-CD11b/c, and PE anti-CD8α (all from BD Biosciences, San Jose, CA). The choice to use CD45RA as a marker for B cells is a unique advantage of working with a rat model where mature B cells can be easily distinguished from other leukocytes on the basis of their expression of CD45 isoforms. Flow cytometry was performed with FACSAria (BD Biosciences) and data were analyzed using FlowJo software (TreeStar, Ashland, OR).
Anti cd4 v450
Manufactured by BD
Sourced in United States
Anti-CD4-V450 is a fluorochrome-conjugated antibody used for the detection and analysis of CD4-positive cells in flow cytometry applications. It binds specifically to the CD4 surface marker, which is expressed on a subset of T lymphocytes. This product is designed for research use only and its performance characteristics have been determined through laboratory testing.
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18 protocols using anti cd4 v450
1
Rat Airway Immune Cell Profiling
2
PBMC Activation, Expansion, and Regulatory T Cell Analysis
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0.5–1.5 × 106 PBMCs were washed twice in PBS then stained with 1.25 μM of carboxyfluorescein succinimidyl ester (CFSE) for 15 min at 37 °C in dark. After five washes in complete medium, cells were incubated with anti-CD2, anti-CD3, and anti-CD28 antibodies from the T cell activation/expansion kit (Miltenyi Biotec) according to the manufacturer’s instructions. At day 3, cells were stained for surface markers (day 3 cells) or cultured for 2 more days in the presence of 100 U/mL of IL-2 (day 5 cells). Staining for CD4 and CD8 and viability dye was carried out for 30 min on ice, using V450 anti-CD4 and BV605 anti-CD8 (BD Biosciences) along with viability dye (LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit, ThermoFisher). For Tregs identification, cells were incubated with antibodies against surface markers (anti-CD25-PE, CD3-FITC, CD127-AF647) for 45 min on ice, washed, fixed/permeabilized, and incubated with CD4-V450 and Foxp3-PE-cy7 (eBioscience). Cells were acquired on the NovoCyte flow cytometer and analyzed using FlowJo v10.
3
Multiparametric Flow Cytometry of PBMCs
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PBMC were thawed and rested overnight at 37°C in RPMI1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100μg/ml streptomycin, and 2 mM glutamine (R10). The following day, the cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen), washed and stained with the following antibodies: anti-CD3-APC-Cy7, anti-CD4-V450, anti-CD8-PE-Cy7, anti-CD45RA-APC (BD Biosience), and anti-CCR7-PE (e-BioScience). The cells were washed and fixed with 1% Formaldehyde in PBS. All data were collected on a BD LSR II flow cytometer (BD Biosience) and analyzed using FlowJo 8.7.7 (TreeStar).
4
Flow Cytometry Analysis of Immune Cell Phenotypes
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The fluorochrome-labeled monoclonal antibodies (mAbs) specific for human monocyte surface and intracellular molecules were used for flow cytometry analysis. The mAbs specific for T cell surface molecules anti-CD14-BV510 (Clone-MφP9), anti-CD3-Alexa-fluor700 (Clone-UCHT1), anti-CD4-PECy7 (Clone-SK3), anti-CD4-V450 (Clone-RPA-T4), anti-CD8-APC (Clone-RPA-T8), anti-TNF-α-PE (Clone-MAb11), anti-IL-6-FITC (Clone-AS12), anti-IL-12-V450 (Clone-C11.5), anti-CD80-FITC (Clone-L307.4), anti-CD81-FITC (Clone-JS-81), Cytofix/Cytoperm and GolgiPlug were obtained from BD Biosciences (San Jose, CA). Anti-CD8-APC-Fluor-780 (Clone-RPA-T8) was purchased from eBioscience (San Diego, CA). Anti-CD40-PercpCy5.5 was purchased from BioLegend (San Diego, CA). Anti-pig SLA-class-I-FITC (Clone-JM1E3) and anti-human CD9-FITC (Clone-MM2/57) were obtained from Bio-Rad (Hercules, CA). Anti-SLA class-II-DR-FITC (clone 2E9/13) was obtained from LifeSpan BioSciences (Seattle, WA). Fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), and RPMI-1640 medium were purchased from Thermo-Fisher Scientific (Waltham, MA). CD14-microbeads, CD4 cell purification kits, and pan-T-cell purification kits were obtained from Miltenyibiotic (Cambridge, MA). Recombinant human tumor necrosis factor alpha (hTNF-α) and interferon gamma (hIFN-γ), and porcine IFN-γ (pIFN-γ) were purchased from R&D System (Minneapolis, MN).
5
Multicolor Flow Cytometry Assay for Murine Immune Cells
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For surface staining, single-cell suspensions were prepared as described before. Staining was performed with Live-Dead viability-staining (propidium iodide or 7-Aminoactinomycin 7AAD (BioLegend) or fixable viability stain 450 (BD Biosciences)). For analysis of the murine cell phenotype, the following antibodies were used:
Anti-CD4-V450 (1:400) (clone: RM4-5, BD Bioscience), Anti-CD4-PeCy7 (1:1000) (clone: RM4-5, BD Bioscience), Anti-CD4-AF700 (1:200) (clone: RM4-5, BD Biosciences), Anti-CD3-APC (1:200) (clone: 1452-C11, BD Bioscience), Anti-IFNγ-V450 (1:200) (clone: XMG1.2, BD Bioscience), Anti-CD45.2- AlexaFluor 700 (1:400) (clone: 104, Thermo Fisher Scientific), Anti-CD45-BV605 (1:200) (clone: 30-F11, BD Bioscience); Anti-CD11b- Pe/Cy7 (1:200) (clone: M1/70, Thermo Fisher Scientific), Anti-CD11b-PerCP-Cy5.5 (1:200) (clone: M1/70, Biolegend), Anti-IL 17-APC (1:200) (clone: eBio17B7, Thermo Fisher Scientific), Anti-CXCR5-FITC (1:200) (clone: L138D7, BioLegend) and Anti-PD-1-PE (1:200) (clone: 29F.1A12, BioLegend), Anti-Bcl6-PECy7 (1:50) (clone: 7D1, Biolegend), Anti-IL21-APC (1:50) (clone: FFA21, Thermo Fisher Scientific).
Flow cytometry was performed with a FACS Canto II57 (link) and data were analyzed using FlowJo 10.0 software (https://www.flowjo.com/solutions/flowjo ).
Anti-CD4-V450 (1:400) (clone: RM4-5, BD Bioscience), Anti-CD4-PeCy7 (1:1000) (clone: RM4-5, BD Bioscience), Anti-CD4-AF700 (1:200) (clone: RM4-5, BD Biosciences), Anti-CD3-APC (1:200) (clone: 1452-C11, BD Bioscience), Anti-IFNγ-V450 (1:200) (clone: XMG1.2, BD Bioscience), Anti-CD45.2- AlexaFluor 700 (1:400) (clone: 104, Thermo Fisher Scientific), Anti-CD45-BV605 (1:200) (clone: 30-F11, BD Bioscience); Anti-CD11b- Pe/Cy7 (1:200) (clone: M1/70, Thermo Fisher Scientific), Anti-CD11b-PerCP-Cy5.5 (1:200) (clone: M1/70, Biolegend), Anti-IL 17-APC (1:200) (clone: eBio17B7, Thermo Fisher Scientific), Anti-CXCR5-FITC (1:200) (clone: L138D7, BioLegend) and Anti-PD-1-PE (1:200) (clone: 29F.1A12, BioLegend), Anti-Bcl6-PECy7 (1:50) (clone: 7D1, Biolegend), Anti-IL21-APC (1:50) (clone: FFA21, Thermo Fisher Scientific).
Flow cytometry was performed with a FACS Canto II57 (link) and data were analyzed using FlowJo 10.0 software (
6
Quantifying SARS-CoV-2 Antigen-Specific T Cell and NK Cell IFN-γ Responses
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The level of IFN-γ secretion by CD4+, CD8+ T cells, and NK cells was measured in whole blood using the following procedures: (1) 100 μl of whole blood was diluted with 400 μl of IMDM medium (Gibico-BRL) and then it was stimulated with Leukocyte Activation Cocktail (BD GolgiPlug™, including 50 ng/ml PMA, 1 μM ionomycin, and 1 μg/ml brefeldin A) or SARS-CoV-2 antigen (0.03 μg/ml RBD, 0.03 μg/ml S1, 0.03 μg/ml S2, and 0.015 μg/ml N) for 4 h at 37 °C with 5% CO2. (2) After stimulation, 200 μl of the supernatant was extracted and incubated at room temperature for 15 min with 5 kinds of antibodies (anti-CD45-APC/H7, anti-CD3-FITC, anti-CD4-V450, anti-CD56-PE/Cy7, and anti-CD8-APC) (BD Biosciences). (3) After the red blood cells were lysed, the cell suspension was fixed and permeated with the Fixation/Permeabilization Buffer at room temperature for 15 min. (4) After washing, the cell suspensions were stained with anti-IFN-γ-PE (BD Pharmingen) at room temperature for 15 min. (5) After washing, the cell pellets were resuspended in 200 μl PBS and analyzed using a flow cytometer (BD Bioscience-Pharmingen).
7
Longitudinal Lymphocyte Phenotyping Analysis
8
Comprehensive Murine Immune Cell Analysis
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Antibodies were purchased as followed: Anti-CD4-V450, anti-CD8α-APCcy7, anti-B220-V450, anti-CD138-Pe-cy7, anti-CD86-Pe-cy5, anti-FAS-PE, anti-GL-7-APC, anti-CD40-APC, anti-MHCII-FITC, anti-IL-4-PE, anti-IL-5-PE, anti-IgM-Pe-cy7, anti-IgG1-APC, anti-IFN-r-Percp5.5, anti-TNF-α-PE, and anti-IL-17-Pe-cy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-PDL-1-biotin (eBioscience, San Diego, CA), anti-XBP-1s (Cell Signaling Technology, Danvers, MA), and anti-Rabbit IgG-FITC (Thermo Fisher Scientific, Waltham, MA) antibodies were purchased from commercial sources. Recombinant mouse IL-4 (PeproTech, Rocky Hill, NJ) and LPS (Sigma-Aldrich, St. Louis, MO) were purchased from the commercial companies. Goat F(ab’)2 Anti-Mouse IgM (1022-01, SouthernBiotech, Birmingham, AL) and anti-mouse CD40 (BE0016-2, BioXCell, Lebanon, NH) were commercially purchased from companies.
9
Comprehensive Flow Cytometry Analysis of Lung and Lymph Node Immune Cells
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BALF cells recovered were labeled with anti‐CD11c‐APC, anti‐Ly6G‐FITC, anti‐CD11b‐PE‐Cy7 (Biolegend), anti‐CD45‐APC‐Cy7, and anti‐Siglec‐F‐PE (BD Biosciences) for 45 min. MLN cells were isolated. The cell clumps were disaggregated into single‐cell suspensions using nylon mesh (70 μm pore size) filtration, and erythrocytes were lysed with RBC lysis. For detection of Th1, Th2, Th17, and Treg cells, the MLN cells isolated above were stimulated with lymphocyte activator mixture for 5 h and labeled with surface markers anti‐CD4‐V450 (BD Biosciences), anti‐CD4‐FITC (eBioscience), or anti‐CD25‐APC (Biolegend). After washing, fixing, and permeabilizing according to the manufacturer's instructions, cells were labeled intracellularly with anti‐IL‐17A‐PE‐Cy7, anti‐Foxp3‐PE (Biolegend), anti‐IFN‐γ‐APC, and anti‐IL‐13‐PE (Thermo Fisher Scientific), and then were incubated for 45 min. After rinsing, all labeled cells were detected using FCM on the FACScan Flow Analyzer. Data were analyzed with FlowJo software.
10
T Cell Differentiation Assay with DC-Derived EVs
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CD4+ naïve T (1.5 × 105 cells/well) isolated by positive selection with anti‐mouse CD4 magnetic beads (R&D Systems) were collected and further cultured with different stimuli‐induced DC‐derived EVs (secreted by 1.2 × 107 cells) for 6 d in 100 μl of T cell medium per well (96‐well plate), and analyzed by FCM. For intracellular cytokine staining, cells were stimulated for 4 h with 1 μg/ml PMA (Sigma‐Aldrich) and 1 μg/ml ionomycin (Calbiochem, Germany) in the presence of 10 μg/ml brefeldin A (Sigma‐Aldrich). Cells were stained for the surface marker anti‐CD4‐V450 (BD Bioscience), anti‐CD4‐FITC (eBioscience, Waltham, MA, USA), and anti‐CD25‐APC (Biolegend). Cells were then fixed, permeabilized and labelled with intracellular and intranuclear staining reagents according to the manufacturer's instructions (eBioscience), and further stained with anti‐IL‐17A‐phycoerythrin (PE)‐cyanine (Cy7), anti‐forkhead box P (Foxp)3‐PE (Biolegend), anti‐IFN‐γ‐APC, or anti‐IL‐13‐PE (Thermo Fisher Scientific). All labeled cells were detected using FCM on the CytExpert Flow Analyzer. Data were analyzed with FlowJo software.
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