The THP-1 cell line was obtained from ATCC (TIB-202), and maintained at 2 × 105 cells per ml in RPMI 1640 medium supplemented with 10% FCS and 2 μM l−1L -glutamine. A total of 2 × 106 THP-1 cells per ml were differentiated using 200 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St Louis, MO, USA) for 72 h. Differentiation of PMA-treated cells was enhanced by changing the PMA-containing media 72 h after the initial treatment with fresh RPMI 1640 (10% FCS and 1% L -glutamine) for 5 additional days.
Phorbol 12 myristate
Manufactured by Merck Group
Sourced in United States, United Kingdom
Phorbol 12-myristate is a chemical compound used in laboratory research. It functions as a protein kinase C activator.
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Lab products found in correlation
Human simian ifn γ kit, by Mabtech (4 mentions)
Ionomycin, by Merck Group (4 mentions)
Tib 202, by American Type Culture Collection (1 mentions)
S mem, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Welgene (1 mentions)
Medium 254, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Rpmi 1640, by Welgene (1 mentions)
Trypsin edta, by Corning (1 mentions)
Lps escherichia coli o111 b4, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Anti vα24 jα18 tcr antibody, by BioLegend (1 mentions)
Krn7000, by Cayman Chemical (1 mentions)
Golgi stop and golgi plug, by BD (1 mentions)
Human il 12, by R&D Systems (1 mentions)
Golgistop, by BD (1 mentions)
Monensin, by Merck Group (1 mentions)
Monensin, by BD (1 mentions)
Anti tnfα clone mp6 xt22, by BD (1 mentions)
16 protocols using phorbol 12 myristate
1
THP-1 Cell Differentiation Protocol
2
Quantifying Mycobacteria-Specific T Cells
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Peripheral blood mononuclear cells were isolated from heparin anti-coagulated blood using standard methods. An IFN-γ ELISpot assay was used to estimate the number of, and IFN-γ production capacity of, mycobacteria-specific T cells in PBMCs using a human/simian IFN-γ kit (MabTech, Nacka. Sweden), as described previously [31] (link). In brief, PBMCs were cultured with 10 μg/ml PPD (SSI, Copenhagen, Denmark), or without antigen, in triplicate, and incubated for 18 h. Phorbol 12-myristate (Sigma-Aldrich Dorset, UK) (100 ng/ml) and ionomycin (CN Biosciences, Nottingham, UK) (1 μg/ml) were used as a positive control. After culture, spots were developed according to the manufacturer's instructions. Determinations from triplicate tests were averaged. Data were analysed by subtracting the mean number of spots in the cells for medium-only control wells from the mean counts of spots in wells with cells and antigen or peptide pools.
3
Murine Melanocyte Cell Culture Protocol
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Melan-a melanocytes are normal, immortalized murine melanocyte cells derived from C57BL/6 mice. Melan-a melanocytes were obtained from Dorothy Bennett (St. George's Hospital, London, UK). Melan-a melanocytes were maintained in a RPMI-1640 (Welgene, Korea) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO). Melan-a Leaden (or Ashen) melanocytes are immortalized Melanophilin (or Rab27a) mutant murine melanocyte cells derived from C57BL/6J 54 . Leaden and Ashen melanocytes were obtained from Dorothy Bennett (St. George's Hospital, London, UK). Cells were maintained in RPMI-1640 (Welgene, Korea) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 200 nM PMA and 2 nM cholera toxin (Sigma-Aldrich, St. Louis, MO). HDFn cells were maintained in Dulbecco's Modified Eagle Medium (Welgene, Korea) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. COS7 monkey kidney cells were maintained in SMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. NHEM cells were maintained in a Medium 254 (gibco) supplemented with 1%HMGS, 100 U/mL penicillin, and 100 µg/mL streptomycin.
4
THP-1 Macrophage Differentiation and Stimulation
5
Quantifying Mycobacterial-Specific T Cells
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An IFN-γ ELISpot assay was used to estimate the numbers and IFN-γ production capacity of mycobacteria-specific T cells in PBMCs using a human/simian IFN-γ kit (MabTech, Nacka. Sweden), as described previously34 (link). PBMCs were cultured with 10 μg/ml PPD (SSI, Copenhagen, Denmark), or a pool containing overlapping 15mer peptides spanning ESAT6 (Peptide Protein Research Ltd, Wickham, UK), or without antigen, in triplicate, and incubated for 18 h. Phorbol 12-myristate (Sigma-Aldrich Dorset, UK) (100 ng/ml) and ionomycin (CN Biosciences, Nottingham, UK) (1 μg/ml) were used as a positive control. After culture, spots were developed according to the manufacturer’s instructions. Spot forming units were counted and average spot areas measured using AID CADAMA ELISPOT reader and software (CADAMA, Stourbridge, UK). Determinations from triplicate tests were averaged. Data were analysed by subtracting the mean number of spots in the wells with cells in medium alone from the mean counts of spots in wells with cells combined with PPD or peptide pools.
6
IFN-γ ELISpot Assay for Mycobacterium-Specific T Cells
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An IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay was used to determine the numbers of IFN-γ producing mycobacterium-specific T cells in PBMCs using a human/monkey IFN-γ kit (Mabtech) as described previously [32 ]. In brief, PBMCs were cultured with 10 μg/mL purified protein derivative (PPD) (SSI, Copenhagen), or without antigen, in triplicate, and incubated for 18 h. Phorbol 12-myristate (100 ng/mL) (Sigma) and ionomycin (1 μg/mL) (CN Biosciences) were used as a positive control. After culture, spots were developed according to the manufacturer’s instructions. Determinations from triplicate tests were averaged. Data were analysed by subtracting the mean number of spots in medium-only control wells from the mean counts in antigen-stimulated wells [31 ].
7
Measuring Secreted GM-CSF from Ocular Tissues
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To measure secreted GM-CSF, full-thickness cornea (two corneas per sample) or bulbar and palpebral conjunctiva (conjunctiva from two eyes per sample) from naïve and DED mice were collected and stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; Sigma-Aldrich Corp.) and ionomycin (500 ng/mL; Sigma-Aldrich Corp.) in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific, Waltham, MA, USA) + 10% FBS for 24 hours at 37°C and 5% CO2. Supernatant was then collected and stored at −80°C with protease inhibitor (Sigma-Aldrich Corp.) until analyzed with a murine GM-CSF ELISA kit (MGM00, R&D Systems, Minneapolis, MN, USA). Samples were run in triplicate.
8
Temporal Kinetics of iNKT Activation
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The iNKT kinetics assay was set up with 2 million PBMCs in 1 ml R10 per condition in a 24-well plate. PBMCs from each donor (n = 4) were stimulated with 1 μg/ml α-Galactosyl Ceramide glycolipid (α-GalCer, also known as KRN7000, Cayman Chemicals, USA). PBMC activation was assessed at 2 h, 4 h, 6 h, 8 h, and 10 h post-stimulation. Further, PBMCs were stimulated for 10 h, centrifuged for 5 min (1500 rpm) and resuspended in R10 for a rest period of 2 h, 4 h and 6 h, respectively. The negative control was an unstimulated ex vivo control and positive control was a 6 h stimulation using Phorbol 12-myristate (PMA—25 ng/ml) and 13-acetate plus ionomycin (Io—500 ng/ml) (Sigma Aldrich, USA). Prior to stimulation, all conditions and controls were pre-stained with the anti-Vα24-Jα18 TCR antibody (6B11, Biolegend, USA), as short-term iNKT stimulation causes iNKT TCR internalization21 (link). Addition of Golgi stop and Golgi plug (BD Biosciences) which allowed intracellular IFN-γ accumulation, were added 2 h, 1 h, and 30 min post-stimulation in the 8 h and 10 h timepoints, 4 h and 6 h timepoints, and 2 h timepoint, respectively. iNKT cell IFN-γ production was the primary outcome. At the end of each timepoint, cells were stained for flow cytometry.
9
NK Cell Stimulation and Activation Assay
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As a control, NK cells (2 × 105) were seeded in 96-well round bottom plates. When indicated, we added 5 ng/mL human IL-12 (R&D Systems) and 20 ng/mL human IL-18 (MBL), or 500 ng/mL ionomycin (Sigma-Aldrich) and 2 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), or K562 tumor cells at a 1:1 effector to target (E:T) ratio. For anti-CD16 mAb, the flat bottom plastic plates (BD Biosciences) were coated with F(ab’)2 anti-mouse IgG (Beckman Coulter) and anti-CD16 (10 µg/mL, 3G8, Beckman Coulter). Cells were stimulated for 4 h in the presence of brefeldin A (Golgistop, BD Biosciences) plus anti-CD107a-FITC and CD107b-FITC monoclonal antibodies, surface stained, and processed for intracellular IFN-γ detection. NK cells are defined as live CD3−CD56+ NK cells.
10
Cytokine Analysis in Murine Spleen Cells
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Spleen cells from different groups of BALB/c mice were analyzed for cytokine content as previously described [48 (link)]. Briefly, T cells were seeded at 106/mL in RPMI 1640–10% FBS, and stimulated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of monensin (BD) during 4-h incubation at 37 °C, and then treated with Cytofix/Cytoperm fixation and permeabilization kit (BD). For macrophage stimulation, spleen cells were seeded at 106/mL in RPMI 1640–10% FBS, and stimulated with LPS 100 ng/mL (Sigma-Aldrich) in the presence of monensin during 4 h incubation at 37 °C. The permeabilized cells were intracellularly stained with the PE-conjugated cytokine-specific mAbs anti-IFNγ (clone XMG1.2), and anti-TNFα (clone MP6-XT22), all from BD; or isotype-matched irrelevant mAbs from BioLegend.
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