PBMNCs or QQMNCs in 10% FBS/RPMI 1640 medium (1×106 cells/mL) were treated with 25 ng/mL of phorbol‐12‐myristate‐13‐acetate (PMA; Promega, Madison, WI) and 1 μg/mL of ionomycin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 12 hours at 37°C. Subsequently, for the last 3 hours, cells were incubated with 2 μmol/L of monensin (BioLegend, San Diego, CA). Thereafter, cells were washed and suspended with 2 mmol/L of EDTA/PBS buffer and stained for cell surface markers with CD4‐PerCP/Cy5.5 and CD25‐PE before fixation. Stained cells were washed, resuspended with 2 mmol/L of EDTA/PBS buffer, and distributed into aliquots for each staining. After treatment with fixation buffer (BioLegend) and permeabilization buffer (BioLegend), cells underwent intracellular staining with interferon‐gamma (INF‐γ)‐Pacific Blue and IL‐4/allophycocyanin (APC). Alternatively, after treatment with the FOXP3 Fix/Perm Buffer Set (BioLegend), cells underwent intranuclear staining with forkhead box protein 3/fluorescein isothiocyanate (Foxp3‐FITC). Intracellular or intranuclear staining was performed, according to the supplemental protocol for each buffer. The cellular frequency of CD4+/INF‐γ+/IL‐4−, CD4+/INF‐γ−/IL‐4+, or CD4+/CD25+/Foxp3+ in CD4+ helper lymphocytes was evaluated as that of T helper (Th)1, Th2, or regulatory T cells.
Phorbol myristate acetate (pma)
Manufactured by Promega
Sourced in United States, United Kingdom
PMA is a lab equipment product manufactured by Promega. It is designed to perform a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or interpretation of the product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (3 mentions)
Anti il 4 antibody, by BioXCell (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Fujifilm (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Permeabilization buffer, by BioLegend (1 mentions)
Foxp3 fix perm buffer set, by BioLegend (1 mentions)
Monensin, by BioLegend (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Diphenyleneiodonium dpi, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Pd98059, by Cayman Chemical (1 mentions)
Hindiii, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Cayman Chemical (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Polymyxin b, by Merck Group (1 mentions)
Rpmi 1640, by Cultilab (1 mentions)
Ecori, by Thermo Fisher Scientific (1 mentions)
10 protocols using phorbol myristate acetate (pma)
1
Th1, Th2, and Treg Cell Profiling
2
Lymph Node Cell Isolation and Stimulation
3
RSV Fusion Protein Characterization
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RSV A2 strain was provided by Dr. Fernando Polack (Vanderbilt University School of Medicine, USA). Human recombinant RSV Fusion protein was purchased from Sino Biological Inc. According to the manufacturer, the glycosylated protein purity is >95% and endotoxin level is <1.0 EU per 1 μg of the protein, as determined by the LAL method. PMA and Protease-free DNase 1 were from Promega. Dextran, LPS O111:B4 from Escherichia coli, Diphenyleneiodonium (DPI), N-acetyl-L-cysteine (NAC), and Histopaque-1077 were obtained from Sigma-Aldrich. ECORI and HINDIII were from Invitrogen. PD98059 and SB203580 were from Cayman Chemical. Polymyxin B and anti-RSV F protein (131-2A) were from Millipore. Blocking anti-TLR-4 (HTA125) and mouse IgG2a isotype control were from eBioscience. The 5-(and-6)-chloromethyl-2’-7’-dichlorodihydrofluorescein diaceate, acetyl ester (CM-H2DCFDA) was from Molecular Probes. RPMI 1640 was from Cultilab, and FCS was from Gibco.
4
Macrophage and PBMC Stimulation Assay
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MΦ (2 × 106 cells per well) and PBMC (1 × 105 cells per well) were plated in a sterile 96-well plate with complete RPMI medium and separately exposed to viable parasites (3 parasites per cell) and stimulated with TbEVs (10 μg·mL−1). PBMC also was stimulated by T. b. brucei soluble antigen (10 μg·mL−1). MΦ plates were incubated for 24 h and PBMC plates for 72 h at 37 °C in a humidified atmosphere with 5% CO2. In parallel, unstimulated cells, used as the negative control, and PMA (Promega, Madison, WI, USA) stimulated MΦ and ConA (Sigma-Aldrich)-stimulated lymphocytes, used as the positive controls, were also incubated.
5
Co-culture of THP-1 Macrophages and PANC-1 Cells
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Human monocytic THP-1 cells maintained in RPMI 1640/10% fetal bovine serum were seeded in 12-well transwell inserts (0.4 μm pore size) at a concentration of 1×105 cells/mL. Settled THP1 cells were treated with 10 ng/mL 12-O-tetradecanoylphorbol-13-acetate (PMA; Promega, Madison, WI, USA, #V1171) for 24 hours in an incubation system (37°C, 5% CO2). The PMA containing media were aspirated gently from the inserts and replaced with media containing with 15 ng/mL lipopolysaccharide (Sigma, St. Louis, MO, USA, #L2630); the cells were incubated for 48 hours to allow differentiation into macrophages. Differentiated macrophages within the inserts were transferred onto preplated human PANC-1 cells (1×105/mL) and cocultured in medium supplemented with 2 μg/mL CD40 antibody (R&D System, Minneapolis, MN, USA, #MAB6321-500) or 2 μg/mL isotype monoclonal antibody control (R&D System, #MAB004) for 96 hours.
6
Hepatocyte-Stellate Cell-Macrophage Coculture
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Huh7.5.1 were a gift from F. Chisari (The Scripps Research Institute, La Jolla, San Diego, California, USA). LX2 were purchased from Merck. THP1 were purchased from ATCC. Huh7.5.1 and LX2 cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% heat-decomplemented FBS, gentamycin (0.05 mg/mL), and nonessential amino acids (complete DMEM) at 37°C with 5% CO2. Cell lines were certified mycoplasma free. For proliferation arrest and differentiation (Huh7.5.1dif cells), Huh7.5.1 cells were cultured in complete DMEM containing 1% DMSO. THP1 cells were cultured and differentiation in RPMI 1640 medium with GlutaMAX-I supplement and HEPES, and they were supplemented with 10% FBS and gentamycin (0.05 mg/mL) (Thermo Fisher Scientific). To generate THP-1–derived macrophages (M0), cells were treated with PMA (320 nM) (Promega) for 48 hours. For coculture experiment, Huh7.5.1 were cultured with 20% LX2 or 20% LX2 and 10% macrophages in complete DMEM for 3 days before treatment.
7
Kallikrein Gene Expression in Activated Monocytes
8
Multiparametric Flow Cytometry of Immune Cells
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Cell suspensions were stained for 25 min with antibodies in PBS with 1% FCS. For flow cytometry analysis, cells were analysed using a BD LSRFortessa™ X-20 (BD Biosciences) or BD LSRII (BD Biosciences) and data were analysed using FlowJo software (Version 10, Treestar Inc). Cells were sometimes fixed in 2–4% paraformaldehyde for FACS analysis. Viability of the cells was determined using the LIVE/DEAD Fixable Blue kit (Life Technologies). Antibodies used include: CD4 (RM4-5; efluor450 (eBioscience)), CD44 (IM7; Percpcy5.5 (eBioscience)), IFNγ (XMG1.2; PE (BD Bioscience)), IL4 (PE, 11B11, eBioscience)), IL5 (APC, TRFK5, BD Bioscience)), IL13 (eFluor660, eBio13A, eBioscience)), IL17a (17B7; PE-Cy7 (eBioscience)), All staining was performed in the presence of FcR Blocking Reagent (Miltenyi Biotec). Intracellular cytokine staining (ICS) was performed following 6 h of re-stimulation with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Promega) and 1 µg/mL ionomycin (Sigma) and BD Golgi Stop and BD Golgi Plug (diluted 1:1000, BD Biosciences). Following surface stain, cells were incubated with eBioscience Fixation/Permeabilization buffer for 25 min followed by 25 min in Permeabilization buffer (eBioscience), and incubation with antibodies in Permeabilization buffer for a further 30 min.
9
Isolation and Stimulation of ILC2s for Naïve T Cell Modulation
10
Isolation and Stimulation of ILC2s for Naïve T Cell Modulation
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Lin−Thy1.2+ KLRG1+ Sca1 + ILC2s were sort
purified from the LP or MLN as described above. Cells were counted, centrifuged
(1,500 r.p.m. for 5 min), and resuspended at a final concentration of 5 ×
104 cells per 50 μl depending on the experiment. Cells
were cultured in cIMDM containing 0.05 mg ml−1 PMA (Promega,
Southampton, UK) and 0.1 mg ml−1 ionomycin (Sigma) for 24 h.
For some experiments, cultures were centrifuged after 3 h and the supernatant
containing PMA and ionomycin was replaced with fresh cIMDM for the remaining 21
h. Supernatants were harvested 24 h poststimulation and stored at − 20
°C. Naïve T cells were FACS purified as CD4+TCRβ+Foxp3-RFP−Il4-GFP−CD25−CD44lowcells from naïve reporter mice. FACS-purified cells were resuspended in
cIMDM at a concentration of 1 × 106/ml. A total of 1 ×
105 naïve T cells were plated onto tissue-culture-treated
flat-bottom 96-well plates coated with CD3 (1 μg ml−1)
and CD28 (10 μg ml−1) antibody at 37 °C for
2–3 h. Cells were pelleted and resuspended in 50 μl ILC2-derived
supernatant with and without the addition of 10 μg ml−1anti-IL-4 antibody (BioXcell).
purified from the LP or MLN as described above. Cells were counted, centrifuged
(1,500 r.p.m. for 5 min), and resuspended at a final concentration of 5 ×
104 cells per 50 μl depending on the experiment. Cells
were cultured in cIMDM containing 0.05 mg ml−1 PMA (Promega,
Southampton, UK) and 0.1 mg ml−1 ionomycin (Sigma) for 24 h.
For some experiments, cultures were centrifuged after 3 h and the supernatant
containing PMA and ionomycin was replaced with fresh cIMDM for the remaining 21
h. Supernatants were harvested 24 h poststimulation and stored at − 20
°C. Naïve T cells were FACS purified as CD4+TCRβ+Foxp3-RFP−Il4-GFP−CD25−CD44lowcells from naïve reporter mice. FACS-purified cells were resuspended in
cIMDM at a concentration of 1 × 106/ml. A total of 1 ×
105 naïve T cells were plated onto tissue-culture-treated
flat-bottom 96-well plates coated with CD3 (1 μg ml−1)
and CD28 (10 μg ml−1) antibody at 37 °C for
2–3 h. Cells were pelleted and resuspended in 50 μl ILC2-derived
supernatant with and without the addition of 10 μg ml−1anti-IL-4 antibody (BioXcell).
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