For Th0 conditions, sorted naïve CD4+ T cells were stimulated (day 0) with plate-bound anti-CD3ε (1 μg/ml; BD Biosciences) and anti-CD28 (3 μg/ml; BD Biosciences) on 48-well-plates (0.3 × 106 cells/well) in 1 ml T cell medium/well (RPMI1640 supplemented with 10% FBS [Sigma/biowest], antibiotics, 50 mM β-mercaptoethanol) supplemented with 20 U/ml recombinant human IL-2 (rhIL-2) (Peprotech) for 3 days, unless otherwise stated. For the assessment of cell proliferation, 1–10 × 106 naïve CD4+ T cells were labeled using Cell Proliferation Dye eFluor™ 450 (Thermo Scientific) according to the manufacturer's protocol prior to activation. Th1 and Th17 cells were generated from sorted naïve CD4+ T cells activated with anti-CD3ε/anti-CD28 for 3 days in the presence of 20 U/ml rhIL-2 (Peprotech), 5 ng/ml IL-12 (R&D Systems) and 3 μg/ml anti-IL-4 (BioXcell) for Th1 cells, and in the presence of 2 ng/ml TGFβ, 10 μg/ml IL-6, 10 μg/ml IL-1α, and 10 μg/ml IL-1β for Th17 cells. For cytokine detection, activated cells were restimulated for 4 h with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) and ionomycin (Iono, 750 ng/ml) (both from Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences).
Ionomycin iono
Manufactured by Merck Group
Sourced in Germany, United States
Ionomycin is a laboratory reagent used as a calcium ionophore. It functions by increasing intracellular calcium levels, which can be useful for various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
Cell proliferation dye efluor 450, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Anti cd3ε, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by BD (1 mentions)
Anti il 4, by BioXCell (1 mentions)
Anti cd3ε, by BD (1 mentions)
Il 12, by R&D Systems (1 mentions)
Resiquimod r848, by InvivoGen (1 mentions)
Cd3 28 beads, by Thermo Fisher Scientific (1 mentions)
Rlt plus buffer, by Qiagen (1 mentions)
Anti cd3 mab, by BD (1 mentions)
Monensin and brefeldin, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Viability stain 510, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Glutamine, by Euroclone (1 mentions)
10 protocols using ionomycin iono
1
Th0, Th1, and Th17 Cell Generation
2
Stimulation and Analysis of Immune Cell Subsets
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Whole PBMC were left untreated or stimulated with 100 ng/ml staphylococcal enterotoxin B (SEB, Sigma) at 2*10e6 cells per ml for 18 h at 37 °C. Supernatants were harvested and stored at −80 °C until use. CD4+, CD8+, CD14+, CD19+, CD56+ sorted cells, and moDCs were either left untreated or stimulated at 2*10e6 cells per ml with 100 ng/ml SEB, 1 μg/ml resiquimod (R848) (InvivoGen)—a toll-like receptor 7 and 8 ligand, a potent activator of both monocytes and B cells—, 10 μ/ml CD3/28 beads (Gibco, Life Technologies) for 18 h or 100 ng/ml phorbol myristate acetate (PMA, Sigma) and 1 μg/ml ionomycin (iono, Sigma) for 6 h at 37 °C.
For mRNA analysis, cells were lysed with RLT Plus buffer (Qiagen) and kept at −20 °C until further extraction.
For mRNA analysis, cells were lysed with RLT Plus buffer (Qiagen) and kept at −20 °C until further extraction.
3
Analyzing CAR T Cell Functionality
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Degranulation, measured by cell surface modulation of CD107a25 (link) and intracellular cytokine production (TNF-α and IFN-γ), was analyzed by flow cytometry of CAR T cells incubated with different target cells. Briefly, CD44v6.CAR T and CD19.CAR T cells from different donors, at day 11–15 after the initial stimulation, were left untreated or stimulated with target cells at the ratio of 1:1. Anti-CD107a Ab (Miltenyi) and monensin and brefeldin (BD Biosciences) were added during the incubation period. As positive control, CAR T cells were stimulated with 10 ng/mL phorbol myristate acetate (PMA; Sigma) and 1 μg/mL ionomycin (IONO; Sigma). After 5 h of incubation, cells were stained with anti-CD3 mAb (BD Bioscience) and Viability Stain 510 (BD Bioscience), fixed, permeabilized (Cytofix/Cytoperm kit, following the manufacturer's instruction; BD Bioscience), and then stained for intracellular cytokines with TNF-α- and IFN-γ (BD Bioscience)-specific Abs. Cells were analyzed by flow cytometry, and viable CD3+/CD4+ or CD3+/CD8+ cells were analyzed for CD107a, TNF-α, and IFN-γ expression. The percentage of positive CAR T cells incubated alone (always <5%) was subtracted to the percentage of positive CAR T cells stimulated with the different targets or PMA/IONO.
4
PBMC Isolation and Cytofluorimetric Analysis
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On day 0 and day 30, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by Ficoll-Hypaque (Eurobio, Les UlisCedex, France) density-gradient centrifugation. Mononuclear cells were resuspended in RPMI (Cambrex BioScience, Verviers, Belgium) completed with 10%heat-inactivated fetal calf serum (FCS, HyClone, South Logan, UT) and 1%glutamine (all from Euroclone, Milan, Italy), and left stimulated with 50 ng/ml Phorbol Myristate Acetate (PMA, Sigma-Aldrich, St Louis, MO), 1 µg/ml Ionomycin (Iono, Sigma-Aldrich). The stimulation was carried out at 37 °C for 5 h, in the presence of the intracellular trafficking inhibitor Brefeldin A (10 µg/ml BFA; Sigma-Aldrich), before intracellular staining and cytofluorimetric analysis.
5
Modulating Signaling Pathways in Cells
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5 µM Brefeldin A (BFA, Sigma-Aldrich) was used to inhibit the secretory pathway and 100 nM Bafilomycin A1 (Baf, SCBT) was used to inhibit lysosomal degradation. To activate PKA signalling, 1 mM dbcAMP (SCBT) was used for 24 h. Wnt signalling was activated by 2 µM WAY-262611 (WAY, SCBT) for 6 h, or by inhibiting GSK-3β with 30 µM BI5521 (opnMe, Boehringer Ingelheim) for 1 h. To modulate Importin-β-dependent transport, 50 µM Ivermectin (ive, SCBT) for 6 h, 0.5 µM ionomycin (iono, Sigma-Aldrich) or 250 nM thapsigargin (thaps, SCBT) for 1 h were used.
6
Activation of Frozen Immune Cells
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Liquid-nitrogen frozen PBMC from healthy donors and from T1D patients were thawed in complete RPMI medium (Gibco RPMI 1640 Medium, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, South Logan, UT, USA), L-glutamine (2mM) (EuroClone S.p.A., MI, Italy) and 1% penicillin/streptomycin (pen/strep) (EuroClone) according to established protocols [19 (link)] and centrifuged at 1200 rpm for 5 minutes at room temperature (RT). Cells were cultured in 48 well plates (Falcon, Corning Incorporated, NY, USA), 1.5×106 cells per well in complete RPMI. Subsequently, cells were stimulated with the addition of 7.5 ng/ml phorbol-12-myristate-13-acetate (PMA) (Calbiochem, Merk, Darmstadt, Germany) and 0.8μg/ml Ionomycin (IONO) (Sigma Aldrich, Merk). The cells were incubated for three to five days at 37°C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, cells were harvested from culture plates and washed by centrifugation 1200 rpm for 5 minutes in PBS at RT. Subsequently cells were stained for FACS analysis as described below.
7
Immune Regulation by PMA, Ionomycin, and OVA
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Phorbol 12-myristate 13-acetate (PMA; #P1585), ionomycin (Iono; #I0634), and OVA (#A5503) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminum hydroxide (alum, #77161) was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and C20 (#S6577) from Selleckchem (Houston, TX, USA). Antibodies against phospho(p)-PKCθ (#9377), p-STAT6 (#5654), STAT6 (#5397), p-NF-κB (#3033), and NFAT (#4389) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-PKCθ (#sc-212), anti-β-actin (#sc-47778), and anti-lamin B1 (#sc-374015) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GATA3 (#ab61052). Anti-inducible nitric oxide synthase (iNOS; #ab136918) antibodies were purchased from Abcam (Cambridge, UK).
8
Macrophage and T Cell Line Protocols
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RAW 264.7 and THP-1
macrophage cells were obtained from the American Type Culture Collection
(ATCC, U.S.A.). Jurkat JE6-1 TLR 4, TLR 6, TLR 2/1, TLR 2/6 and TLR
2/1/6 were a kind gift from Peter Steinberg’s Lab (Medical
University of Vienna). Hank’s balanced salt solution (HBSS),
Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park
Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), phorbol
12-myristate 13-acetate (PMA) and ionomycin (iono) was purchased from
Sigma-Aldrich. Trypsin-ethylenediaminetetraacetic acid (EDTA) was
purchased from Invitrogen, U.S.A.
macrophage cells were obtained from the American Type Culture Collection
(ATCC, U.S.A.). Jurkat JE6-1 TLR 4, TLR 6, TLR 2/1, TLR 2/6 and TLR
2/1/6 were a kind gift from Peter Steinberg’s Lab (Medical
University of Vienna). Hank’s balanced salt solution (HBSS),
Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park
Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), phorbol
12-myristate 13-acetate (PMA) and ionomycin (iono) was purchased from
Sigma-Aldrich. Trypsin-ethylenediaminetetraacetic acid (EDTA) was
purchased from Invitrogen, U.S.A.
9
Cytomegalovirus-specific T Cell Assay
10
Tacrolimus Effects on T-cell Activation
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