MCF-7 cells were seeded in 96-well plates overnight and then transfected with pGL4.10-ckβ(−2000/−1) and pGL4.73[hRluc/SV40]. Six hours after transfection, the culture medium was replaced with 1% (v/v) serum-free DMEM for 20 hr before stimulation. The cells were treated with 10, 20, or 30 ng/mL PMA (EMD Chemicals, USA) for 6 hr, except for the time-course study, in which cells were treated with 20 ng/mL PMA for 6 to 48 hr. For controls, DMSO was added to the cells instead of PMA. For the identification of PKC isozyme involved in the PMA-mediated repression of the ckβ promoter, MCF-7 cells were individually or co-treated with PMA (20 ng/mL) and a PKC inhibitor [1 mM PKC412 (Tocris Bioscience, USA), 0.1 mM Go 6983 (Tocris Bioscience, USA), 10 µM PKCε inhibitor peptide (Santa Cruz, USA) or 10 µM PKCη pseudo-substrate inhibitor (Santa Cruz, USA)] for 6 hr. After the treatments, luciferase activities were measured using the Dual-Glo luciferase assay. To determine whether the PMA response element is localized to the Ets and GATA binding sites, pGL4.10-mut(Ets), pGL4.10-mut(GATA), and pGL4.10-mut(Ets/GATA) mutant constructs were individually transfected into MCF-7 cells, followed by incubation with 20 ng/mL PMA for 6 hr before luciferase activity was determined.
Phorbol myristate acetate (pma)
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
The PMA is a lab equipment product manufactured by Bio-Techne. It is a device that measures the production of various cytokines and chemokines in cell culture supernatants. The PMA utilizes a multiplex assay format to allow for the simultaneous quantification of multiple analytes from a single sample.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Bio-Techne (3 mentions)
G 6983, by Bio-Techne (3 mentions)
Gf109203x, by Bio-Techne (2 mentions)
Go6983, by Bio-Techne (2 mentions)
Recombinant murine il 23, by BioLegend (2 mentions)
Ionomycin, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Enzyme linked immunosorbent assay, by BD (1 mentions)
Histopaque, by Merck Group (1 mentions)
Anti cd28, by BD (1 mentions)
Bryostatin 1, by Bio-Techne (1 mentions)
Cocl2, by Santa Cruz Biotechnology (1 mentions)
Bay 87 2243, by Selleck Chemicals (1 mentions)
Bay11 7082, by Santa Cruz Biotechnology (1 mentions)
U0126, by Selleck Chemicals (1 mentions)
Pd0325901, by Selleck Chemicals (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
17 protocols using phorbol myristate acetate (pma)
1
PMA-Mediated Regulation of ckβ Promoter
2
T Cell Activation and Culture Protocol
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Vero E6 (provided by Wendy Maury, PhD, University of Iowa), Vero (American Type Culture Collection), Ramos-B (American Type Culture Collection), and Jurkat E6.1 T cells were maintained in medium, as described elsewhere [16 ]. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donors, as described elsewhere, or from the University of Iowa DeGowin Blood Center and were prepared using Histopaque (Sigma-Aldrich) purification [17 (link)]. CD3+ or CD4+ T cells were further purified (>90%) from PBMCs by negative selection (R&D Systems), with purity assessed by means of flow cytometry (Supplementary Figure 1 ). Jurkat cells were activated with 1-µg/mL anti-CD3 (Invitrogen) and anti-CD28 (BD Biosciences) or 50-ng/mL PMA (Tocris Bioscience) and 1-µg/mL ionomycin (Alfa Aesar). Primary cells (1 million cells/mL) were activated with 200-ng/mL anti-CD3 (Invitrogen) or 5-ng/mL PMA and 100-ng/mL ionomycin. Cell activation was assessed by measuring interleukin 2 release 16 hours after stimulation, using enzyme-linked immunosorbent assay (BD Biosciences) as described elsewhere [18 (link), 19 (link)].
3
Regulatory Transcription Factors Modulation
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The HIF1α activator CoCl2 and the NF-κB inhibitor Bay 11-7082 were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The MEK inhibitors PD0325901 and U0126 as well as the HIF1α inhibitor Bay 87-2243 were purchased from SelleckChem (Munich, Germany). The PKCα activators PMA, bryostatin 1, and PDBu were purchased from Tocris Biosciences (Bristol, UK).
4
Flow Cytometry Immune Cell Profiling
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Antibodies (Ab, see Supplementary Data 4 ) to mouse CD19, CD5, CD11b, CD43, 4-1BBL, IFNγ, IL1β, TGFβ, IL10, IL6, CD11b, and CD45 and their isotype-matched control Ab were purchased from Biolegend, eBioscience, BD Bioscience, and R&D Systems, unless specified. For IC cytokine staining, cells were stimulated with 50 ng/ml PMA (Tocris Bioscience) and 500 ng/ml ionomycin (Tocris Bioscience) for 1–2 h, followed by Golgi stop for 3–4 h using 10 μmol/l of Monensin or Brefeldin A (eBioscience); and then stained following manufacturer’s instruction for IC fixation and permeabilization (eBioscience). Concentration of antibody used in FACS staining was 1 μg per 106 cells. Data were analyzed on FACS Canto II (BD) or CytoFLEX (Beckman Coulter, Inc.) using FlowJo software (Tree Star, Inc.) or CytExpert software (Beckman Coulter, Inc.).
5
Ovarian Cancer Cell Line Maintenance and Stimulation
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The ovarian cancer cell lines SKOV-3, CAOV-3, OVCA433, RMUG-L and ES-2 (obtained from the Cell Bank of the Chinese Academy of Science, Shanghai, China) were maintained at 37°C in a humidified 5% CO2 atmosphere in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco, Invitrogen, Carlsbad, CA), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). The cells were serum starved through incubation in serum-free medium for 12–24 hours before the start of the experiments. β-Acetyl-γ-O-alkyl-L-α-phosphatidylcholine (PAF), epidermal growth factor (EGF), WEB2086 (PAFR antagonist), AG1478 (EGFR inhibitor) and PP2 (Src inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO). U73122 (PLC inhibitor), BAPTA-AM (calcium chelator), Thapsigargin (Ca2+-ATPase inhibitor), GF109203X (PKC inhibitor), and PMA (PKC activator) were obtained from Tocris (Bristol, UK). The rabbit polyclonal antibodies used in this study were directed against phospho/total-EGFR, phospho/total-ERK, and phospho/total-Src. All antibodies were purchased from Cell Signaling Technology (Boston, MA). The mouse monoclonal antibodies used in this study were directed against actin (Sigma, Missouri, USA).
6
Cerebellar Slice Culture Preparation
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Slice cultures were prepared as described previously (Gugger et al., 2012 (link)). Briefly, mice were decapitated at postnatal day 8 (P8), their brains were aseptically removed, and the cerebellum was dissected in ice-cold preparation medium: MEM, 1% Glutamax (Invitrogen), pH 7.3. Sagittal sections (350 µm thickness) were cut on a McIllwain tissue chopper under aseptic conditions. Slices were separated, transferred onto permeable membranes (Millicell-CM, Millipore), and incubated on a layer of neurobasal medium: Neurobasal A medium (Invitrogen) supplemented with B27 supplement (Invitrogen) and Glutamax (Invitrogen), pH 7.3, in a humidified atmosphere with 5% CO2 at 37°C. The medium was changed every 2-3 d; 300 nm PMA (Tocris Bioscience) was added for PKC activation 24 h before slices were fixed; 10 μm Gö6983 (Tocris Bioscience) was added to the medium at each medium change for PKC inhibition, starting at 3 DIV (DIV3). Slices were kept in culture for a total of 7 d and analyzed with Western blot and immunohistochemical staining. To monitor protein degradation, slices were treated with 50 µg/ml cycloheximide (Sigma Millipore) or 10 μm MG132 (Sigma Millipore) or both at DIV7, and protein was extracted at DIV8.
7
Neuronal Signaling Pathway Modulation
8
Chemical Reagents for Cell Signaling
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PMA was purchased from Tocris Bioscience; bisindolylmaleimide I (BIS I), BIS V, and calyculin A from EMD Biosciences; rapamycin from LC Laboratories; PAR2 activating peptide (AP; N-SLIGKT-C) from United Biosystems Inc.; Compound101 (Lowe et al., 2015 (link)) from Hello Bio Limited; and Trypsin (T8802) from Sigma-Aldrich. The stocks of PMA (1 mM), compound101 (30 mM), BIS I (1 mM), BIS V (1 mM), rapamycin (5 mM), and calyculin A (1 mM) were dissolved in DMSO. AP (10 mM) and trypsin (500 µM) were reconstituted into distilled water.
9
PBMC Cytokine and Checkpoint Profiling
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We cultured PBMCs in 48-well plates and added Phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, TOCRIS, USA) plus ionomycin calcium salt (1 μM/ml, Sigma-Aldrich, USA) for 16 h. We added protein transport inhibitor (BD Bioscience, USA) to the co-culture 12 h before we stopped the stimulation. After coculturing, PBMCs were stained for CD4, CD8, interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and TIM3 expression. The cytokine and immune checkpoint expressions were measured by flow cytometry and were analyzed in BD FACSuite V software (BD, Biosciences, USA).
10
Pharmacological Modulation of Ion Currents
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All drugs were purchased from Sigma (MO, USA) unless otherwise indicated. Stock solutions of 4-aminopyridine (4-AP), pertussis toxin (PTX), cholera toxin (CTX), PMA (Tocris Bioscience, Ellisville, MO) and ω-conotoxin MVIIC (Tocris Bioscience, Ellisville, MO) were prepared in distilled deionized water. Z941 was a kind gift from Dr. Terrance P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada). Stock solutions of cholecystokinin-8 (Tocris Bioscience, Ellisville, MO), LY294002, CCK-4, nifedipine, forskolin, gallein, wortmannin, KT5720 (RD system), devazepide, LY225910, GW5823, BC264, SP600125, SB203580, anisomycin, PP2, PP3, Akt inhibitor III, U0126, and GF109203X were prepared in dimethylsulfoxide (DMSO). The final concentration of DMSO in the bath solution was estimated to be less than 0.01%, and this compound had no functional effects on IA (not shown).
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