LPLs and pLN cells stimulated for 4 hours with PMA (50 ng/ml; Sigma), ionomycine (1 μg/ml; Sigma), and the Golgi-traffic inhibitor Brefeldin (1 μl/ml; BD Biosciences). Cells were stained with anti-CD3-PE (BD Pharmingen) or anti-CD3-APC (eBioscience) and anti-CD4-APC (Biolegend) or anti-CD4-FITC (BD Pharmingen). Next, the cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience). For intracellular staining the cells were incubated in permeabilization buffer (eBioscience) containing anti-IL-17-FITC (Biolegend), anti-IFNγ-FITC (BD Pharmingen), anti-IL-4-PE (BD Pharmingen) or Foxp3-FITC (eBioscience). An appropriate isotype matched control antibody was used in all FACS analyses. Cells were analyzed on a FACS Calibur using the CellQuest software (BD Biosciences). Results were analyzed with FlowJo version 7.6.5.
Ionomycine
Manufactured by Merck Group
Sourced in United States
Ionomycine is a laboratory reagent used in cell biology research. It functions as a calcium ionophore, facilitating the movement of calcium ions across cell membranes. This property enables researchers to investigate calcium-dependent cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (7 mentions)
Brefeldin a, by Merck Group (4 mentions)
Anti il 4 pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Ebio13a, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions)
Trfk5, by BD (2 mentions)
Brefeldin, by BD (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3 apc, by Thermo Fisher Scientific (1 mentions)
Anti il 17 fitc, by BioLegend (1 mentions)
Anti ifn γ fitc, by BD (1 mentions)
Foxp3 fitc, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd4 apc, by BioLegend (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Anti cd3 pe, by BD (1 mentions)
Foxp3 kit, by Thermo Fisher Scientific (1 mentions)
Brefeldin a bfa, by Merck Group (1 mentions)
14 protocols using ionomycine
1
Characterization of Th17 and Treg Cells
2
Phenotyping of immune cell subsets
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Flow cytometry was performed to analyze the phenotype and frequency of various subpopulations in TILs, NILs and PBMC using a series of fluorescence labeled monoclonal antibodies specific for CD3, CD4, CD8, CD25, CD56, CTLA-4, CD45RO, CD127, αβTCR, TCR Vα24, γδTCR, IFN-γ, perforin with appropriate isotype-matched controls. Anti-TCR Vα24-FITC was obtained from Beckman Coulter (Beckman Coulter Inc, Brea, CA). Antibodies against CD45RO, CTLA-4, and CD127 were purchased from eBioscience (San Diego, CA), and the rest from BD Pharmingen (San Diego, CA). PBS57 loaded and unloaded control mCD1d tetramers were kindly provided by the tetramer facility of NIH. Staining for FOXP3 protein was performed using the FOXP3 kit (eBioscience) according to the manufacturer’s instructions. For intracellular staining of IFN-γ and perforin, cells were first stimulated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, Saint Louis, MO) and 1 μg/ml ionomycine (Sigma-Aldrich) for 4 hours with the addition of 10 μg/ml brefeldin A (BFA, Sigma-Aldrich) in the last 2 hours. After surface staining, cells were fixed and permeabilized using CytoFix/Cytoperm (BD Pharmingen), followed by staining for intracellular antigens. Data were acquired using FACSCalibur (BD Biosciences) and analyzed using the CellQuest software (BD Biosciences, San Diego, CA, USA).
3
NK Cell-Mediated K562 Cell Cytotoxicity Assay
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A total of 1 × 106 expanded NK cells are incubated for 6 h at 37 °C, 5% CO2, 95% RH with cells from the myeloid cell line K562 (gift from Dr. J.J. Bosch, Department of Medicine 5, University Hospital Erlangen) at an NK-to-K562 cell ratio of 20:1 and 5:1 in a final volume of 500 μl cRPMI supplemented with anti-CD107a antibody (clone H4A3, 10 µl/ml, BD Biosciences). K562 cells are confirmed negative for mycoplasma contamination. To prevent protein secretion and degradation of internalized CD107a, monensin (1 µM) and brefeldin A (10 ng/ml, both from Sigma) are added after 1 h of incubation. NK cells alone serve as a negative control, and NK cells stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and ionomycine (250 ng/ml, both from Sigma) serve as a positive control for anti-CD107a antibody binding. After 6 h of incubation, cells are harvested, washed, resuspended in 50 μl PBS, and stained with live–dead Zombie NIR (BioLegend), anti-CD56 (clone CHD56, BioLegend), and CD16 antibody (clone 3G8, BioLegend). Samples are analyzed using a Becton Dickinson FACS CANTOII flow cytometer and Flowjo software.
4
Analyzing Th Cell Populations in SMCs
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Freshly isolated SMCs were activated with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, USA) and ionomycine (Sigma-Aldrich, USA) after having been cultured with or without resveratrol for 2 h. The cells were then stained with anti-CD4-PerCP, anti-IL-4-PE (BD Biosciences, USA), anti-IFN-γ-APC (BD Biosciences, USA), and anti-IL-17-Alexa Fluor 488 (BD Biosciences, USA) to investigate Th1, Th2, and Th17+ cell populations by flow cytometry.
5
Intracellular Cytokine Profiling of CD4+ T-cells
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Intracellular flow cytometry was utilized to determine the percentage of IFN-γ, IL-4, IL-17 and IL-10-producing lymphocytes within the CD4+subset in 10 patients, in both IC and PB samples. Briefly, heparinized whole blood was diluted 1:1 with RPMI medium (Sigma-Aldrich Co., St Louis, MO, USA), and cells were stimulated for 5 h at 37 °C, in 5 % CO2 atmosphere with 0.5 ng/ml ionomycine (Sigma-Aldrich) and 1 γg/ml phorbol myristate acetate (PMA; Sigma-Aldrich) in the presence of 5 mg/ml Brefeldin A (Sigma-Aldrich) to induce cytokine production (Grille et al. 2010 (link)). Lymphocytes were then stained with anti-human CD4 and anti-human CD3 (BD Pharmingen, San Diego, USA). Red blood cells were lysed with FACS Lysing Solution (BD Biosciences), and lymphocytes were permeabilized with FACS Permeabilizing Solution (BD Biosciences). To detect intracellular cytokines, cells were stained with the following cytokine-specific antibodies: anti-human IL-17-PE, IFN-γ-APC, IL-4-PE and IL-10-APC (BD Pharmingen, San Diego, USA), washed, and fixed with 1 % paraformaldehyde. Data acquisition and analysis was performed as previously described. A gate was set on CD4+CD3+ lymphocytes and at least 5000 cells were counted and evaluated for the expression of IFN-γ, IL-17, IL-4 and IL-10.
6
PBMC Proliferation Assay for Vaccine Response
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PBMCs (1 × 105/well) were incubated in triplets at 37 °C and 5% CO2 in 96-well round bottom plates with the aluminum hydroxide-free and human albumin-free TBE antigen (FSME Ticovac strain Neudörfl, Pfizer, 0.6 µg/mL), the CEF MHC-I control peptide pool (Anaspec, 32 peptides, 2.5 µg/mL per peptide), a positive control peptide mix consisting of CEFT MHC-II peptide pool (JPT Peptides, 14 peptides, 0.25 µg/mL per peptide, each corresponding to a defined HLA class II restricted T-cell epitope from cytomegalovirus (CMV), Epstein–Barr virus (EBV), influenza virus or Clostridium tetani), tetanus toxoid (Statens Serum Institute, Copenhagen, Denmark, 0.125 Lf/mL) and tuberculin purified protein derivative PPD (Statens Serum Institute, Copenhagen, Denmark, 0.5 µg/mL); further, a mix consisting of phorbol myristate acetate (Sigma, 1.25 × 10−8 M) and ionomycine (Sigma, 0.1 µg/mL) or medium alone (RPMI, HyClone plus 2% Human Serum, Sigma). After 120 h cells were pulsed with methyl-[3H]thymidine (1 µCi/well) for 18 h and T-cell proliferation was quantified on a MicroBeta2 Microplate Counter (PerkinElmer, Waltham, MA, USA) as counts × 1000/min. Data was then standardized based on unstimulated controls of each patient.
7
Flow Cytometry Immunophenotyping and Cytokine Analysis
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Surface staining was conducted in phosphate-buffered saline (PBS) for 15 min at room temperature. Intracellular staining was conducted by fixing cells with Cytofix/Cytoperm buffer (15 min, +4°C) and then staining with the antibody in Perm/wash buffer overnight at +4°C (BD Biosciences, Franklin Lakes, NJ, USA). For cytokine evaluation, the cells were stimulated in complete RPMI 1640 medium with phorbol myristate acetate (500 ng/ml; Sigma-Aldrich, St. Louis, MI, USA), ionomycine (1.4 μg/ml; Sigma-Aldrich), and GolgiStop (BD Biosciences). Zombie Aqua and Zombie ultraviolet fixable viability markers (BioLegend) were used to gate on living cells. Flow cytometry acquisition was conducted with Cytoflex (Beckman Coulter, Brea, CA) and cell sorting using FACSAria (BD). For cell sorting, the purity of the sorted population was over 95%. Flow cytometry analysis was conducted using FlowJo software (BD Biosciences).
8
Flow Cytometric Analysis of Regulatory T Cells and Bregs in EAE Mouse Model
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When PLP139–151 immunized mice developed EAE with a score of 1, they were randomized to receive plain water or water containing 0.25 mg/ml homotaurine for 5 days. Splenic mononuclear cells (106/tube) from the control and homotaurine-treated groups were treated with 1 µg anti-CD16/anti-CD32 and stained with FITC-anti-CD8α, APC-anti-CD122 and PE-anti-PD-1. After being washed, the frequency of CD8+CD122+PD1− and CD8+CD122+PD-1+ regulatory T cells was determined by flow cytometry. In addition, splenic mononuclear cells (106/tube) were stained with FITC-anti-CD4, fixed, permeabilized and intracellularly stained with PE-anti-Foxp3. The frequency of CD4+Foxp3+ Tregs was determined by flow cytometry. Moreover, splenic mononuclear cells (106/tube) were stimulated with 50 nM PMA and 1 µM ionomycine (Sigma) for 2 h and in the presence of monensin for another 3 h. The cells were stained with FITC-anti-CD19, fixed, permeabilized and intracellularly stained with PE-anti-IL-10. The cells with isotype controls served as the negative controls. The frequency of CD19+IL-10+ Bregs was determined by flow cytometry.
9
Quantifying Antigen-Specific Immune Responses by ELISPOT
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The enzyme-linked immunospot (ELISPOT) is a sensitive technique for the detection of cytokineproducing cells at the single cell level. The IFN-γ ELISPOT (Diacole) method was used for measuring the antigen-specific response to EV-CPC elicited by NK cells, T cells, antigen-specific Th1 CD4 + and CD8 + effector T cells. Primed splenocytes of each vaccinated mouse were isolated, plated (at a seeding density of 1.5 million cells/cm 2 in 96-well plates), and re-exposed to either EV (2x10 9 particles) or PBS in vitro for 21h; the positive control was a mixed solution of phorbol 12myristate 13-acetate (PMA, 100ng/mL) and ionomycine (2ug/mL; both from Sigma) that triggers unspecific IFN-γ production. When spots indicating IFN-γ production appeared, they were counted using a Cellular Technology Ltd system (USA) and analyzed using ImmunoSpot 5.0.3 software.
Results are reported as the mean of triplicate measurements of the number of IFN-γ spot-forming cells (SFCs) per 5x10 5 plated cells.
Results are reported as the mean of triplicate measurements of the number of IFN-γ spot-forming cells (SFCs) per 5x10 5 plated cells.
10
Activating Immune Cells for Intracellular Analysis
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Single cell suspensions were stimulated during 4 h using 1 µg/ml ionomycine (Sigma-Aldrich, USA) and 5 ng/ml PMA (Calbiochem, USA) in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich, USA). Cells were harvested and stained by standard intracellular staining procedures (see above).
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