Freshly isolated cells or CD4 single-positive T cells purified by the FACS Aria II cell sorter (BD Biosciences) from TET tissues were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), 1 µg/ml ionomycin (Sigma-Aldrich), and Golgi Stop reagent (BD Biosciences) at 37 °C, 5% CO2, for 5 hours. Harvested cells were washed and stained with antibodies against surface antigens and fixable viability dye (eBioscience) at 4 °C for 20 minutes. After the incubation, cells were washed, fixed, and permeabilized with Cytofix/Cytoperm solution (BD Bioscience) at 4 °C for 30 minutes. Intracellular cytokines (IFN-γ, TNF-α, and IL-2) were then stained with antibodies for IFN-γ (clone 4SB3; Biolegend), TNF-α (clone MAb11; Biolegend), and IL-2 (clone MQ1-17H12; eBioscience), followed by FACS analyses.
Golgi stop reagent
Manufactured by BD
Sourced in United States
Golgi-stop reagent is a cell culture supplement that disrupts protein transport from the endoplasmic reticulum to the Golgi apparatus. It is commonly used in cell biology research to inhibit the secretory pathway and accumulate proteins within the cell for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (6 mentions)
Cd11b, by BD (4 mentions)
Cd103, by BD (4 mentions)
Ifn γ, by Thermo Fisher Scientific (4 mentions)
Siglec f, by BD (4 mentions)
Il 17a, by BD (4 mentions)
Facscanto 2, by BD (3 mentions)
Penicillin, by Merck Group (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Interleukin 2 (il 2), by BD (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Staining buffer, by BD (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Anti cd45, by BD (2 mentions)
Collagenase a, by Roche (2 mentions)
Mhcii, by BD (2 mentions)
Ionomycin, by BD (2 mentions)
Jagged1, by BioLegend (2 mentions)
28 protocols using golgi stop reagent
1
Cytokine Profiling of T Cells
2
Intracellular Cytokine Staining and Analysis
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Immunofluorescent staining was conducted as previously described (Lee et al., 2010 (link)). For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of the Golgi-stop reagent (BD Biosciences). After they were fixed with 4% paraformaldehyde and permeabilized with 1% saponin, the cells were intracellularly stained with FITC-conjugated anti-IL17A, PE-conjugated anti-IL-4, and APC-conjugated anti-IFN-γ antibodies. Data were acquired with the FACS Canto II instrument (BD Biosciences) and analyzed with the FlowJo V10 software. Cytokine levels were measured by the BD CBA Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) in accordance with the manufacturer’s instructions.
3
NK Cell Degranulation Assay with PBMC
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On day 6 PBMC, either GAD65 AA 114–122 or FLU peptides stimulated for 4 days, additionally cultured for two days in the presence of IL-2 (100 IU/ml), were co-cultured with pulsed or unpulsed APCs at 1:3 (PBMC:APCs) ratio in 96 well round bottomed culture plates (Corning Incorporated, Corning, NY 14831–001, USA) for 2 and half hours in RPMI 10% FBS complete medium (see above) additionally supplemented with GolgiStop reagent (1:500 dilution, BD Biosciences). An experimental positive control was set-up by NK cell isolation from PBMC of a HD volunteer previously obtained with the RosetteSep method (Stem Cell Technologies, Vancouver, Canada) and FicollPaque Plus (Lympholyte, Cedarlane Laboratories, Burlington, Ontario, USA). Isolated NK cells have been routinely checked for the percentage of CD3-CD56+ cells by FACS analysis and those with purity greater than 90% were cultured with 200 IU/ml of recombinant human IL-2 (Sigma-Aldrich) at 37°C and used up to 5 days after isolation as effectors in degranulation assay. Isolated NK cells were then co-cultured with K562 cells (American Type Culture Collection, ATCC), a tumoral cell line known to induce NK cell degranulation according to standard protocols, as control target [41 (link)], and with either GAD65 AA 114–122 peptide pulsed and unpulsed APCs.
4
Spleen Cell Cytokine Analysis by Flow
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Single-cell suspensions of spleens were prepared and assayed by flow cytometry as described30 (link). The fluorochrome-conjugated monoclonal Abs used are listed in Supplementary Table 1 . To detect cytokine expression, splenocytes were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μM ionomycin (Sigma-Aldrich) in the presence of Golgi-stop reagent (BD Biosciences) for 5 h and treated with Cytofix/Cytoperm Fixation/Permeabilization Solution (BD Biosciences).
5
Isolation and Cytokine Expression Analysis of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples collected in tubes with EDTA by conventional ficoll-uropoline density gradient centrifugation. PBMCs were then washed with phosphate-buffered saline (PBS) and resuspended in RPMI1640 medium supplemented with 5% FBS, penicillin (100 U/mL-streptomycin (100 μg/mL), and 2-mercaptoethanol (5 × 10−5 M) (all purchased from Sigma Aldrich, Saint Louis, MO, USA). The expression of intracellular TNF-α, IL-6, and IFN-γ was studied in PBMCs cultivated for 48 h in vitro (5 × 105/0.5 mL) in the presence of IL-2 (100 U/mL) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/mL) Sigma-Aldrich, Saint Louis, MO, USA), or PMA (50 ng/mL) with ionomycin (500 ng/mL) (Sigma Aldrich, Saint Louis, MO, USA). Control cells were left without stimulation. 5 h before the end Golgi Stop reagent (0.5 μL/well in 0.5 mL of medium, BD Biosciences, San Jose, CA, USA) was added to PBMC cultures to stop extracellular export of cytokines. Then, PBMCs were collected and washed with 1 mL of BD Staining Buffer. Cell viability of PBMCs was monitored with trypan blue exclusion assay.
6
Assessing T Cell Cytokine Production
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Following co-culture of γδ and CD4+ T cells with autologous macrophages or osteoclasts for 3 days, T cells were harvested and stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin in the presence of Golgistop reagent (BD Biosciences) for a further 6 h. γδ T cells and CD4+ T cells were then harvested and stained with anti-human γδ-TCR-FITC or anti-human CD4-FITC, respectively, prior to fixation and permeabilisation with a Cytofix/Cytoperm kit (BD Biosciences). T cells were then stained using a monoclonal mouse anti-human IFNγ-V450 antibody or mouse IgG1, κ-V450 isotype control (both BD Biosciences), and monoclonal mouse anti-human-IL-17-PE or mouse IgG1-PE isotype control (both eBiosciences). IFNγ- and IL-17-producing T cells were then assessed using flow cytometric analysis with an LSR II flow cytometer, and data were analysed with FlowJo software.
7
NK Cell Activation Assay
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Each mouse was injected with 200 μg poly I:C (P9582, Sigma, MO, US) 18 h prior to harvest to prime the NK cells. The primed splenic NK cells (2 × 106) were cocultured for 4 h with the same number of RMA-S (the murine RMA-S mutant cell lines have a defect in class I assembly and express markedly reduced levels of class I molecules at the cell surface) and YAC-1 (target of NK cells, murine T cell lymphoma, have defects in MHC class I expression) tumor cells. Monoclonal antibodies against CD107a and IFN-γ or the respective isotype controls were subsequently added. The GolgiStop™ reagent (554724, BD Biosciences, CA, US) was added to inhibit the secretion of intracellular CD107a and IFN-γ. Medium was used as the negative control. After stimulation, cells were harvested, washed with PBS, and subjected to FACS to detect the expression of IFN-γ and CD107a.
8
PBMC Activation and Cytokine Expression
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Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples collected in tubes with EDTA by conventional ficoll-uropoline density gradient centrifugation. PBMCs were then washed and resuspended in RPMI1640 medium supplemented with 5% FBS, penicillin (100 U/ml) – streptomycin (100 μg/ml) and 2-mercaptoethanol (5 × 10− 5 M) (all purchased from Sigma - Aldrich, Saint Louis, MO, USA). Cells (5 × 105 / 0.5 ml) were cultured for 48 h in the absence (control) or presence of IL-2 (100 U/ml) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/ml) or PMA (50 ng/ml) and ionomycin (500 ng/ml, all purchased from Sigma-Aldrich). PBMCs treated in this way were studied for the expression of SIRT1, SOD2 and HSP70 (surface and intracellular). The intracellular expression of TNF and IFN-γ, was studied in PBMCs (5 × 105 / 0.5 ml) cultured in the absence (control) or presence of IL-2 (100 U/ml) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/ml) (Sigma-Aldrich, Saint Louis, MO, USA) or PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma - Aldrich, Saint Louis, MO, USA) for 5 h. Simultaneously, Golgi Stop reagent (0.5 μl / well in 0.5 ml of medium, BD Biosciences, San Jose, CA, USA) was added to PBMC cultures (5 × 105 / 0.5 ml) to stop extracellular export of cytokines. Then PBMCs were collected and washed with 1 ml of BD Staining Buffer.
9
Isolation and Analysis of Murine Lung Cells
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Collagenase digestion of mouse lung tissue was previously described in 30 (link). In short, mice were euthanized by CO2 asphyxiation after which perfusion was carried out by injecting 3–5 mL of phosphate buffered saline (PBS) through the right ventricle of the heart. Lungs were resected, minced with scissors, and digested in complete DMEM containing 15 mg/mL collagenase A (Roche, Indianapolis, IN), and 2500 units of DNase I (Sigma-Aldrich, St. Louis, MO) for 30 m at 37°C. Digested tissue was disrupted through a 10 mL syringe, filtered through a 100 μM pore size nytex screen and centrifuged through a 20 % Percoll solution in serum free media. 10x106 cells were then stained with appropriately diluted fluorophore-conjugated antibodies for 30 m. For intracellular cytokine staining (ICS) cells were diluted to 1x106/mL and stimulated for 4 h with PMA (10 ng / mL), ionomycin (10 μM), and Golgi-stop reagent (BD Bioscience, San Jose, CA). Antibodies used in this study are as follows: anti-CD45, CD11b, CD4, IL-17A, MHCII (I-Ab), CD103, SiglecF (BD Bioscience, San Jose, CA), CD11c, IFNγ (eBioscience San Diego, CA), DLL1, DLL4, Jagged1, Jagged2, and CD64 (Biolegend, San Diego, CA).
10
Multiparametric Flow Cytometry of Immune Cells
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Single cell suspensions of spleen and BM were prepared and assayed by FACS as described (42 (link)). To detect cytokine expression, CD4+ T cells were stimulated with 20 ng/ml PMA and 1 μM ionomycin (both from Sigma-Aldrich) in the presence of Golgi-stop reagent (BD Biosciences). For intracellular staining, cells were treated with Fixation/Permeabilization Solution (BD Biosciences). We used monoclonal antibodies (mAbs) to CD45 (30-F11), Ter119 (Ter119), CD11b (M1/70), Gr-1 (RB6-8C5), Ly6G (1A8), Ly6C (HK1.4), B220 (RA3-6B2), F4/80 (BM8), CD16/32 (2.4G2), CD34 (RAM34), Sca-1 (D7), CD117 (2B8), Flt3 (A2F10.1), CD4 (RM4-5. GK1.5), CD3 (145-2C11), CD8 (53-6.7), ICOS (C398.4A), BCL6 (K112-91), CD115 (T38-320), BAFF (121808), CD224 (A-4), MHC II (M5/114.15.2), CD80 (16-10A1), CD86 (GL1), CD138 (281-2), GL7 (GL7), FAS (Jo2), IFN-γ (XMG1.2), GM-CSF (MP1-22E9), IL-6 (MP5-20F3), IgG1 (A85-1; Santa Cruz), and MCL1 (REA924; Myltenyi Biotec), rat IgG2a (RTK2758), 7-aminoactinomycin D (7-AAD), and Annexin V (All from eBioscience or BD Biosciences unless indicated). Abs were conjugated with FITC, PE, PE-cy7, PerCP, allophycocyanin, or allophycocyanin-Cy7. Data were acquired with a FACS Canto II (BD Biosciences) and analyzed with Flowjo software (Tree Star Inc).
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