Splenic lymphocytes were stained for surface markers as follows. For extracellular markers, single cells were stained at 2 × 106 cells per well in a 96-well V bottomed plate. T cells were stained with anti-CD3 Alexa 700 (BD, clone 500A2), anti-CD4 PerCP (BD, clone RM4-5), anti-CD44 FITC (BD, clone IM7), anti-CD62L V450 (BD, clone MEL-14), and anti-FOXP3 APC (BD, clone MF23). B cells were stained with anti-CD19 PEcy7 (eBiosciences, clone 6D5), anti-B220 FITC (eBiosciences, clone RA3-6B2), anti-CD21 APC (BD, clone 7G6), anti-CD23 PE (BD), and anti-CD80 V450 (BD, clone 16-10A1). Fifty microliters of the antibody master mix prepared in MACS buffer (1× PBS, 2 mM EDTA, and 0.5% BSA) was added per well in all staining procedures. Cells were acquired on an LSRII (Becton Dickinson) and analyzed by FlowJo (Tree Star, Ashland).
Anti cd19 pecy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CD19-PECy7 is a fluorochrome-conjugated antibody that binds to the CD19 cell surface antigen. CD19 is a transmembrane protein expressed on B cells and B cell precursors. The PECy7 fluorochrome label allows for the detection and analysis of CD19-positive cells using flow cytometry.
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Lab products found in correlation
Anti b220 fitc, by Thermo Fisher Scientific (2 mentions)
Anti cd8 apc efluor780, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 fitc, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Anti cd44 fitc, by BD (1 mentions)
Anti cd3 alexa 700, by BD (1 mentions)
Anti cd23 pe, by BD (1 mentions)
Anti foxp3 apc, by BD (1 mentions)
Anti cd21 apc, by BD (1 mentions)
Anti cd80 v450, by BD (1 mentions)
Anti cd62l v450, by BD (1 mentions)
Anti cd4 percp, by BD (1 mentions)
Anti cd11b apc, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Anti cd4 percp, by Thermo Fisher Scientific (1 mentions)
Anti mhcii fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd3 apc, by BD (1 mentions)
Anti b220 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd4 apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Anti cd38 alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti cd19 pecy7
1
Multiparametric Analysis of Splenic Lymphocytes
2
Multicolor Flow Cytometry of Mouse Immune Cells
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Freshly isolated liver MNCs and spleen cells were initially incubated with anti-mouse CD16/32 (1:100 final dilution; Becton Dickinson) for 15 minutes at room temperature. The washed cells were incubated for 30 minutes at 4 °C with the following fluorescently labeled monoclonal antibodies: anti-ST2L-PE (eBioscience, San Diego, CA), anti-CD11b-APC (eBioscience), anti-CD19-PE-Cy7 (eBioscience), anti-CD4-PerCP (eBioscience), anti-CD3-APC (BD Pharmingen, San Diego, CA), anti-F4/80-FITC (eBioscience), anti-F4/80-PerCP-Cy5.5 (eBioscience), anti-MHCII-FITC (eBioscience). FACS analysis was performed using FACScalibur and/or FACSverse (both from Becton Dickinson).
3
Multiparametric Flow Cytometry of Immune Cell Subsets
4
Phenotyping of Immune Cell Subsets
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After thawing PBMCs in PBS containing the endonuclease benzonase (Novagen/Merck Milipore Schwalbach, Germany), cells were directly stained in PBEA buffer (PBS, 0.5% BSA, 2 mM EDTA and 0.01% NaN3 ) for 30 min on 4°C. An overview of the B cell-, T cell- and NK cell/regulatory T cell (Treg)-phenotyping panels including fluorochromes is provided in Table S2. Cells were washed with PBEA buffer and fixed in PBS containing 1% formaldehyde. The following monoclonal antibodies were used after initial testing and titration: anti-CD3 Alexa-eFluor 780, anti-CD4 PE-Cy7, anti-CD19 Pe-Cy7, anti-CD27 APC, anti-CD28 FITC, and anti-CCR7 PE (eBioscience, SanDiego, USA), anti-CD4 PerCP-Cy5.5, anti-CD8+ PerCP-Cy5.5, anti-CD16 PerCP-Cy5.5, anti-CD20 FITC, anti-CD25 V450, anti-CD38 PerCP-Cy5.5, anti-CD45RA HV450, anti-CD69 FITC, anti-CD86 V450, anti-IFN-g APC, anti-IgD PE, anti-TNF-a FITC (Becton Dickenson GmbH, Heidelberg, Germany), anti-IL-2 PacificBlue (BioLegend,); anti-CD56 PE (Miltenyi, Bergisch Gladbach, Germany).
5
Quantitative Analysis of Immune Cells in BAL
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BAL were obtained and analyzed one day after the last challenge (7 to 9 mice per group). One mL of PBS was instilled intra-tracheally until lungs are inflated uniformly and then slowly aspirated and centrifugated. Total cell number was determined on Kova slide® by optical microscopy. Differential cell counts are determined by flow cytometry with flow cytometer BD LSR II after staining with anti-CD3 APC, anti-CD19 PE-Cy7, FITC anti-F4/80 (eBiosciences, paris, France), anti-CCR3 PE (R&D, Lille, France), anti-PerCP-Cy5.5 Ly6G, anti-CD8 APC-H7 (BD Biosciences, Le Pont-de-Claix,France) and DAPI. Leukocyte populations were identified as neutrophils (Ly6G high), macrophages (F4/80 high DAPI mid (autofluorescence)), eosinophils (CCR3 high), B cells (CD3 négative CD19 high), T cells (CD3 high CD19 négative), and the proportion of CD8+ T cells is determined by the CD8 high population.
6
Intracellular IFN-γ Production in T Cells
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To analyze intracellular IFN-γ production by CD8+ and CD4+ T cells, splenocytes from the mice vaccinated with 1, 3, and 5 doses of cGM-CSF were collected 7 days after the last immunization and stimulated in vitro with 10 µg/mL HPV-16 E7 MHC class I peptide (aa 49–57, RAHYNIVTF) or MHC II (aa 44–60, QAEPDRAHYNIVTFCCK) peptide by incubation at 37 °C with 5% CO2 for 15 h. After 15 h, the cells were treated with 50 ng/mL phorbol-12-myristate-13-acetate (PMA) and 1 µg/mL of ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop protein transport inhibitor containing monensin (BD Bioscience, San Jose, CA, USA) for 4 h. The cultured cells were analyzed by flow cytometry.
For IKDC analysis, splenic cells were stained with anti-B220-FITC (BD Bioscience, San Diego, CA), anti-NK 1.1-perCP (eBioscience, San Diego, CA, USA), anti-TCRβ-PE (eBioscience, San Diego, CA, USA), anti-CD19-PEcy7 (eBioscience, San Diego, CA, USA), and anti-CD11c-APC antibodies (Biolegend, San Diego, CA, USA) at 4 °C for 20 min followed by flow cytometric analysis.
For IKDC analysis, splenic cells were stained with anti-B220-FITC (BD Bioscience, San Diego, CA), anti-NK 1.1-perCP (eBioscience, San Diego, CA, USA), anti-TCRβ-PE (eBioscience, San Diego, CA, USA), anti-CD19-PEcy7 (eBioscience, San Diego, CA, USA), and anti-CD11c-APC antibodies (Biolegend, San Diego, CA, USA) at 4 °C for 20 min followed by flow cytometric analysis.
7
FACS and PLA Analysis of Mouse and Human B Cells
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For FACS analysis of mouse spleen B cells or transfected TKO cells, following fluorophore- conjugated anti-mouse antibodies were used: Anti-CD45R-PerCP-Cy5.5 (RA3-6B2), Anti-IgM-APC, IgM-PE, IgM-FITC (II/41; all eBioscience, Frankfurt, Germany), Anti-CD20-APC, Anti-CD19-PE-Cy7, anti-IgD-AF647, Anti-IgD-APC, Anti-IgD-PE (11-26c.2a; all eBioscience).
For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience). Igα (HMK7/A9; abcam, Cambridge, UK), Syk (Syk-01; BioLegend, San Diego, CA).
For PLA probes against human BCR, the following unlabelled antibodies were used: IgD (IA6-2; BioLegend), IgD (IADB6) and IgM (SA-DA4) from Acris Antibodies (Herford, Germany), and IgM (Fc5u) from Genway Biotech (San Diego, CA).
For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience). Igα (HMK7/A9; abcam, Cambridge, UK), Syk (Syk-01; BioLegend, San Diego, CA).
For PLA probes against human BCR, the following unlabelled antibodies were used: IgD (IA6-2; BioLegend), IgD (IADB6) and IgM (SA-DA4) from Acris Antibodies (Herford, Germany), and IgM (Fc5u) from Genway Biotech (San Diego, CA).
8
Comprehensive Murine Immune Cell Profiling
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Mouse cells were preincubated with purified antimouse CD16/CD32, and human cells were incubated with FcR-blocking reagent (BD Biosciences). Isolated cells were stained with labeled antibodies in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA). Dead cells were excluded based on staining with Live/Dead fixable dye (eBioscience). Forkhead box P3 (FOXP3) fixative solution (eBioscience) was used for FOXP3 staining. Prepared samples were analyzed using a flow cytometer (FACSCalibur or FACSAria; BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The following beads and antibodies were used: OneComp eBeads (eBioscience), antimouse CD11c Brilliant Violet 421 (BioLegend), antimouse CD11b PE-cy7 (eBioscience), GR1-APC (eBioscience), LY6C-APC-eFluor (eBioscience), F4/80 PE (eBioscience), NK-1.1 Percp-Cy5.5 (eBioscience), anti-CD45 FITC (eBioscience), Live/Dead Aqua (eBioscience), anti-CD4 eFluor 450 (eBioscience), anti-CD8 APC eFluor 780 (eBioscience), anti-CD3 Percp-Cy5.5 (eBioscience), anti-CD19 PE-CY7 (eBioscience), anti-CD25 APC (eBioscience), anti-Foxp3 PE (eBioscience), and anti-CD3 APC (BioLegend).
9
Exercise-Induced Changes in Lymphocyte Phenotypes
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Peripheral blood samples were obtained by venous puncture before exercise (baseline period, T0), at exercise exhaustion (post-exercise period, T1), and after resting one hour (resting period, T2). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected in EDTA tubes by Ficoll-Hypaque density gradient centrifugation, followed by cryopreservation in fetal calf serum with 10 % dimethyl sulfoxide until time of analysis. Flow cytometry of samples was analyzed using a previously published methodology (Martínez-Rodríguez et al., 2011) , staining samples with the following monoclonal antibodies: anti-CD3-PerCP, anti-CD56-BV510, anti-CD8-Alexa Fluor 700, anti-CD4-PECy7 (BD Pharmingen), and anti-CD19-PECy7 (eBiosciences). NK cells were defined as CD3-CD56+ cells, identifying CD56 dim and CD56 bright NK cell subsets based on the staining intensity of the specific CD56 monoclonal antibody; T cells and B cells were defined as CD3+ and CD3-CD19+ lymphocytes, respectively. Chemokine receptors expression by lymphocytes were assessed using the following monoclonal antibodies: anti-CXCR5-BV421, anti-CX3CR1-BV650, anti-CXCR3-BV711, anti-CCR7-AF647, anti-CCR1-PE, and anti-CCR5-BV785 (Biolegend). Samples were acquired in full spectrum Cytek Aurora Analyzer (Cytek Biosciences, Fremont, USA), analyzing data using FlowJo software (tree Star, Oregon, USA).
10
Stimulation and Analysis of Mouse T Cells
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Mouse cell suspensions were stimulated with 50 μg/mL of TT or HEL peptide in the presence of BD GolgiPlug™ (BD Biosciences) for 2 h (37 °C). Cells were fixed and stained using the BD Cytofix/Cytoperm™ Kit (BD Biosciences). Surface and intracellular staining (ICS) were performed according to the manufacturer’s instructions, with anti-CD4-FITC, anti-CD3-APC-eFluor®780, anti-CD19-PECy7, anti-TNF-α-PerCP-e710, anti-IL-2-APC and anti-IFNγ-EF450 from eBioscience and anti-CD8a-PE from BD Biosciences.
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