PBMC were activated and stained according to the manufacturer’s instructions (BD Biosciences, Mountainview, CA). Detailed methods are provided in the (S1 Methods ). For identification of Th1 and Th2 cells, or Th17 and Treg cells, four-color antibody cocktails (Th1/Th2/Th17 phenotyping kit, Treg/Th17 phenotyping kit, BD Biosciences) were used. For identification of IL-10+ and TGFβ+ cells, anti-CD4 PerCP-Cy5.5, anti-TGFβ1 PE, and anti-IL-10 APC (all BD Biosciences) were used. anti-CD4 PerCP-Cy5.5, PE-conjugated mouse IgG1κ and APC-conjugated mouse IgG2aκ (all BD Biosciences) were used as isotype control. All samples were analyzed on a CyAn ADP flow cytometer (Beckman Coulter, Lane Cove, NSW, Australia) using Summit software (version 4.3.2, Beckman Coulter). At least 100,000 gated events were collected for each sample. Postacquisition analysis was performed using FlowJo software (version 9.5.2, Tree Star, Ashland, OR). Representative gating and fluorescence histograms are shown in (S1 Fig .). CD4+ values were expressed as a percent of total lymphocytes; all other subsets as a percent of CD4+ cells.
Anti cd4 percp cy5
Manufactured by BD
Sourced in United States
Anti-CD4-PerCP-Cy5.5 is a fluorescent-labeled monoclonal antibody that specifically binds to the CD4 antigen expressed on the surface of T helper cells. It is designed for use in flow cytometry applications for the identification and enumeration of CD4-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (5 mentions)
Ionomycin, by Merck Group (5 mentions)
Anti cd8 apc h7, by BD (4 mentions)
Anti cd62l apc, by BD (4 mentions)
Anti ifn γ apc, by BD (4 mentions)
Anti cd3 apc, by BD (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (4 mentions)
Anti cd8 fitc, by BD (3 mentions)
Anti il 2 pe, by BD (3 mentions)
Anti cd3 pe cy7, by BD (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Facscanto 2, by FlowJo (3 mentions)
Anti cd25 pe, by BD (3 mentions)
Anti cd3 apc cy7, by BD (3 mentions)
Anti cd127 pe, by BD (2 mentions)
Anti il 17 apc, by Thermo Fisher Scientific (2 mentions)
Anti il 22 pe, by R&D Systems (2 mentions)
Anti il 4 pe, by BD (2 mentions)
Anti cd3 fitc, by BD (2 mentions)
39 protocols using anti cd4 percp cy5
1
PBMC Phenotyping for Th1, Th2, Th17, and Treg
2
Multiparametric Flow Cytometry Analysis
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Freshly isolated responders or the generated cells were analyzed with flow cytometry on days 0, 7, and 14. For the analysis of lymphocyte and monocyte lineage markers, the following conjugated antibodies (Abs) were used: anti-CD4-PerCP Cy5.5, anti-CD8-APC H7, anti-CD3-HV450, and anti-CD19-HV500 (all from BD Biosciences, San Jose, USA); and anti-CD16-PE, anti-CD56-PE, anti-CD14-PC7, and anti-CD45-APC AF700 (all from Beckman Coulter, Marseille, France). CD subsets were determined using the Lineage Cocktail 1 (lin 1; FITC-conjugated Abs against CD3, CD14, CD16, CD20, and CD56; all from BD Biosciences), anti-CD123-PECF594, anti-CD11c-PC7 (both from BD Biosciences), and anti-HLA DR-APC (Beckman Coulter). Tregs were identified using anti-CD127-PE, anti-CD4-PerCP Cy5.5 (BD Biosciences), anti-CD25 PC7 (Biolegend, San Diego, USA), and anti-FOXP3-AF647, clone 236A/E7 (BD Biosciences). The staining procedure, including fixation and permeabilization, was performed according to the manufacturer’s instructions (eBiosciences, San Diego, CA, USA). To prevent non-specific binding, mouse immunoglobulin G1 was added prior to incubation with the FOXP3 antibody. The data were acquired with a Navios flow cytometry instrument and analyzed using Kaluza software (Beckman Coulter, Brea, USA).
3
Flow Cytometry Analysis of Splenocyte Activation
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Based our protocol from previous publication (16 ), we resuspended the splenocytes from the immunized in 10μl of RPMI-1640 media. The cells were ex vivo stimulated with anti-CD107a antibody (BD Biosciences, 553792), anti-CD28/CD49d antibody (BD Biosciences, 347690) and OLP or DMSO in 10μl. The mixture was incubated in 5% CO2, 37°C incubator for 1hour and treated with 4μl of mixture of the complete RPMI-1640 media: Brefeldin A (GolgiPlug, BD Biosciences, 555029):Monensin (GolgiStop, BD Biosciences, 554715) in 55:3:2. After 12hours of incubation in 5% CO2, 37°C, the cells were washed with PBS and stained with surface antibodies (anti-CD44 BV421 [BD Biosciences, 536970], anti-CD8a BV510 [BD Biosciences, 563068], anti-CD62L BV650 [BD Biosciences, 564108], anti-CD3 BV786 [BD Biosciences, 564010], anti-CD4 PerCP-Cy5.5 [BD Biosciences, 561115], and anti-CD19 APC [BD Biosciences, 561738]) for 15 minutes at RT and washed. Cells were then fixed with 4% paraformaldehyde in PBS and permeabilized. We then washed the cells and stained with FACS antibodies (anti-IL-2 FITC [BD Biosciences, 562040], anti-TNF PE [BD Biosciences, 554419], anti-IFN-γ [BD Biosciences, 557735], and anti-CD107a [BD Biosciences, 560647]) by 20 minutes of incubation at room temperature, washed twice with the permeabilization buffer and resuspended with 300μl of PBS.
4
Multi-Marker Immunophenotyping of T Cells
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Cells were stained with anti-CD3-eF650 (Clone: UCHT1; eBioscience), anti-CD4-PerCP/Cy5.5 (Clone: SK3; BD Bioscience), anti-CD8-BV570 (Clone: 3B5; Invitrogen), and Live/Dead NearIR (Invitrogen). The surface staining was performed in PBS for 15 min at room temperature in the dark and subsequently fixed using Permeabilizing Solution 2 (BD Biosciences) for 10 min at room temperature.
Intracellular staining was performed with anti-CD137-PE/Cy5 (Clone: 4B4–1; BD Biosciences), anti-CD154-APC/Cy7 (Clone: 24–31; Biolegend), anti-PD1-BV710 (Clone: EH12.2H7; Biolegend), and anti-TIM3-APC (Clone: F38-2E2; eBioscience) for 30 min at room temperature in the dark.
Samples were acquired on a LSRII-Fortessa (BD Biosciences) and at least 1.5 × 106 events were recorded. Calibration of the instrument was reconfirmed weekly with rainbow beads (BD Biosciences).
Intracellular staining was performed with anti-CD137-PE/Cy5 (Clone: 4B4–1; BD Biosciences), anti-CD154-APC/Cy7 (Clone: 24–31; Biolegend), anti-PD1-BV710 (Clone: EH12.2H7; Biolegend), and anti-TIM3-APC (Clone: F38-2E2; eBioscience) for 30 min at room temperature in the dark.
Samples were acquired on a LSRII-Fortessa (BD Biosciences) and at least 1.5 × 106 events were recorded. Calibration of the instrument was reconfirmed weekly with rainbow beads (BD Biosciences).
5
Isolation and Phenotypic Analysis of Colonic Mononuclear Cells
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Mucosal mononuclear cells were isolated from colonic biopsy specimens by collagenase type II (Sigma, Hamburg, Germany) digestion as described previously2 (link),3 (link). The following antibodies were used for phenotypic analysis: anti-CD3-allophycocyanin (APC), anti-CD3-peridinin chlorophyll protein Cy5.5 (PerCPCy5.5; both clone SP34; BD Biosciences), anti-CD4-PerCPCy5.5, anti-CD4-APC (both L200; BD), anti-CD8-phycoerythrin (PE), anti-CD8-fluorescein (FITC; both DK25; Dako), anti-CD45RA-FITC (ALB11; Beckman Coulter), and anti-CCR5-PE (3A9; BD). Lymphocytes were gated on the basis of characteristic forward and side scatter properties. CD4+ T cells were identified by co-expression of CD3 and lack of CD8 expression and memory CD4+ T cells were identified by lack of CD45RA expression on CD4+ T cells. The total number of analyzed colonic cells was between 360,000 and 550,000 and cells in the CD3+ T cell gate consisted of 14,000 – 44,000 colonic cells. CD4+ T cell counts in fresh whole blood were quantified by the use of Trucount Tubes (BD) according to the manufacturer’s instructions. Data were acquired on the FACS Calibur (BD) and analyzed with FlowJo software version 8.8.4. (BD).
6
Tetramer-Based Identification of Antigen-Specific CTLs
7
Multiparametric Flow Cytometry Analysis
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Spleen cell suspensions from each mouse were obtained by dissociation of tissue and red blood cells were lysed with 0.85% ammonium chloride solution. To analyze T cells subsets, one million splenocytes were stained for 15 minutes with the following antibodies: anti-CD3-BV605, anti-CD4-PerCP-Cy5.5, anti-CD44-PE-Cy7 and anti-CD62L-APC (BD Biosciences). After incubation, the cells were washed with PBS and fixed with 1% formaldehyde in PBS. To analyze Treg cells, 1X106 splenocytes were stained with anti-CD3-BV421, anti-CD4-APC and anti-CD25-APC-Cy7. To detect intracellular FoxP3 expression, cells were permeabilized (Cytofix/Cytoperm TM Fixation/Permeabilization Solution Kit, BD Biosciences) and anti-FoxP3-Alexa488 was added and incubated for 30 minutes. Data were acquired in BD LRS Fortessa flow cytometer (Becton-Dickinson, San Jose, CA) and analyzed by FlowJo software (FlowJo, Tree Star, Ashland, OR).
8
Flow Cytometry Staining Reagents
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Anti-CD3 FITC, anti-CD3 APC, anti-CD4 PerCP-cy5.5, anti-CD8-PerCP-cy5.5, anti-CD8 FITC, anti-CD45RO FITC, anti-CD45RO PE, anti-IFN-γ FITC, anti-IFN-γ APC, anti-IL-4 PE and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-22 PE was purchased from R&D Systems (Abingdon, UK). Anti-IL-17 APC was purchased from eBioscience (San Diego, CA, USA). Phorbol myristate acetate (PMA), ionomycin, saponin and Brefeldin A (BFA) were purchased from Sigma-Aldrich (Fluka, Sigma, USA).
9
Multi-Marker Phenotyping of T Cells
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Anti-CD4 PerCP, Anti-CD4 PerCP-cy5.5, anti-CD3 APC, anti-CXCR5-Alexafluor 488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, anti-IL-21 PE, anti-IL-4 PE, anti-IFN-γ APC and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-17 APC and ELISA for determining IL-21 was purchased from eBioscience (San Diego, CA, USA). Anti-IL-22 PE was purchased from R & D Systems (Abingdon, UK). PMA, ionomycin (INO), saponin and Brefeldin A were purchased from Sigma-Aldrich (Fluka, Sigma, USA).
10
Cytokine Profile of Antigen-Specific T Cells
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1.0 × 106 PBMCs were stimulated with 1 μg/ml mesothelin peptide pool (Peptides & Elephants, Potsdam, Germany), medium control or PMA/Ionomycin (positive control, Sigma-Aldrich, St Louis, MI; USA) in R10 medium in the presence of Brefeldin A (10μg/mL, Sigma-Aldrich St Louis, MI, USA) for 6 hours at 37°C. Stimulation was stopped by transferring the cells to a 4°C refrigerator, followed by washing with FACS buffer and staining with the following reagents: anti-CD3 Pacific blue (BD Biosciences, CA, USA), anti-CD4 PerCP-Cy5.5 (BD Biosciences, CA, USA) and anti-CD8 APC-Cy7 (BD Biosciences, CA, USA). After a 15-minute incubation at 4°C, the cells were washed and fixed with a Fix/Perm reagent (Beckman coulter, CA, USA), followed by a further 30-minute incubation at 4°C with an intracellular antibody mix (anti-TNFα APC (BD Biosciences CA, USA), anti-IFN-γ PE-Cy7 (BD Biosciences CA, USA), anti-IL-2 PE (BD biosciences CA, USA) and anti-IL-17 FITC (BioLegend, CA, USA)). The stained cells were then washed with FACS buffer and acquired on a FACSAria flow cytometer (BD Biosciences, Stockholm, Sweden). Data was analysed using FlowJo software (Treestar Inc.).
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