A total of 1 × 106 MC were cultured in 24-well plates and stimulated with either 100 ng/mL Pam2Cys (EMC microcollections, Tübingen, Germany), 100 ng/mL Pam3Cys (EMC microcollections, Tübingen, Germany), 10 ng/mL LPS from s.minnesota (Alexis, Lausen, Swiss), 0.5 µM CpG OD 1668 (Eurofins MWC Operon, Ebersberg, Germany), 10 ng/mL PolyIC (Invivogen, Toulouse, France), 10 ng/mL imiquimod (Invivogen), 10 ng/mL IL-33 (PeproTech, Hamburg, Germany) ionomycin (Sigma-Aldrich/Merck) or phorbol-12-myristat-13-acetate (PMA) (Sigma-Aldrich/Merck) for 24 h at 37 °C in MC-medium. Supernatants were quantified by enzyme-linked immunosorbent assay for IL-10 (R&D Systems, Minneapolis, MN, USA, detection limit 15.6–1000 pg/mL), IL-6 (R&D Systems, Minneapolis, MN, USA, detection limit 15.6–1000 pg/mL), TNF-a (R&D Systems, detection limit 10.9–700 pg/mL), LegendPlex Mouse Inflammation panel (BioLegend, San Diego, CA, USA) and LegendPlex Mouse Th Cytokine panel (Biolegend).
Phorbol 12 myristat 13 acetate pma
Manufactured by Merck Group
Sourced in France
Phorbol-12-myristat-13-acetate (PMA) is a chemical compound commonly used as a laboratory tool. It functions as a potent activator of protein kinase C, a family of enzymes involved in various cellular signaling pathways.
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Lab products found in correlation
Ionomycin, by Merck Group (2 mentions)
Legendplex mouse th cytokine panel, by BioLegend (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Il 33, by Thermo Fisher Scientific (1 mentions)
Legendplex mouse inflammation panel, by BioLegend (1 mentions)
Imiquimod, by InvivoGen (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
Transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
Guava system, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Brefeldin a, by BD (1 mentions)
Phorbol myristate acetate (pma), by R&D Systems (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
6 protocols using phorbol 12 myristat 13 acetate pma
1
Cytokine Production by Murine Immune Cells
2
Multiparametric Flow Cytometry Analysis
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Single-cell suspensions were incubated with the Fc-receptor blocking reagent (Miltenyi Biotec) and then stained with immunofluorescence-conjugated mAbs following the manufacturer’s instructions (Table S5). For transcription factor staining, the cells were first surface stained before fixation and permeabilization using the transcription factor staining kit (eBioscience), followed by intranuclear staining. For intracellular cytokine staining, CD8+ T cells (1.0 × 105 cells) were stimulated in vitro with 50 ng/mL Phorbol-12-myristat-13-acetate (PMA, Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) for 5 h in the presence of 5 μg/mL GolgiPlug. Cell surface molecules were stained before fixation and permeabilization, using the fixation/permeabilization kit (BD Biosciences), followed by intracellular staining. Cell viability was determined using the Annexin V apoptosis detection kits (BioLegend), according to manufacturer’s instructions. The cell samples were analyzed on a flow cytometer (FACS Canto II or Accuri C6; BD Biosciences), and data were analyzed using FlowJo software v10.7 (Tree Star Inc.).
3
Brain Lymphocyte Isolation and Analysis
4
Macrophage Polarization Using THP-1 Cells
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Nonadherent THP-1 cells (ATCC® TIB-202™) were grown in an RPMI 1640 medium (Roswell Park Memorial Institute) supplemented by 10% v/v of fetal calf serum (FCS) uncompleted (Sigma-Aldrich, St Quentin Fallavier, France), 1% v/v antibiotics (penicillin at 100 U·mL−1 et streptomycin at 100 µg·mL−1, Sigma-Aldrich) and 2-mercaptoethanol for a final concentration of 0.05 nM (Sigma-Aldrich).
THP-1 monocytes were differentiated into adherent macrophages (M0) by adding 320 nM phorbol-12-myristat-13-acetate (PMA) (Sigma-Aldrich). At 24 h post-PMA incubation, the culture medium was changed by adding 100 ng·mL−1 lipopolysaccharide (LPS, R&D systems, Minneapolis, MN, USA) and 20 ng·mL−1 interferon gamma human recombinant (IFN-γ, R&D systems) for an M1 polarization. For M2, 20 ng·mL−1 interleukine-4 human recombinant (IL-4, R&D systems) and 20 ng·mL−1 interleukine-13 human recombinant (IL-13, R&D systems) were added. After 72 h, the cells were used for experiments.
THP-1 monocytes were differentiated into adherent macrophages (M0) by adding 320 nM phorbol-12-myristat-13-acetate (PMA) (Sigma-Aldrich). At 24 h post-PMA incubation, the culture medium was changed by adding 100 ng·mL−1 lipopolysaccharide (LPS, R&D systems, Minneapolis, MN, USA) and 20 ng·mL−1 interferon gamma human recombinant (IFN-γ, R&D systems) for an M1 polarization. For M2, 20 ng·mL−1 interleukine-4 human recombinant (IL-4, R&D systems) and 20 ng·mL−1 interleukine-13 human recombinant (IL-13, R&D systems) were added. After 72 h, the cells were used for experiments.
5
Spatiotemporal Regulation of Cell Signaling
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Morpholinos were obtained from Gene-Tools and dissolved in distilled water. Morpholinos (MOs) targeting cdkn1a/p21 (3 ng/embryo) (Sidi et al., 2008) , vegfab (5 ng/ embryo) (Bahary et al., 2007) and a standard control MO (Hasan et al., 2017) have been described previously. To inhibit PI3K, embryos were dechorionated and incubated in 10 uM LY-294002 hydrochloride (SIGMA) for 10 hours prior to imaging. To specifically inhibit PI3K 110 alpha, embryos were dechorionated and incubated in 10 uM, 25 uM or 50 uM of GDC-0326 (Cayman Chemical). To activate ERK signalling, embryos were dechorionated and incubated in 0.25 uM Phorbol-12myristat-13-acetate (PMA) (SIGMA) for 4 hours prior to imaging.
6
Chemical Procurement for Research
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Most standard chemicals were purchased from Sigma (St. Louis, MO), Roche Molecular Biochemicals (Mannheim, Germany), or Merck and Co. (Darmstadt, Germany) unless otherwise indicated. Bromodeoxyuridine (BrdU) and phorbol 12-myristat 13-acetate (PMA) were from Sigma (Lisle d'Abeau Chesne, France). G € o6976 was from Merck Chemistry (Fontenay-sous-Bois, France). AVP and dDAVP were from Bachem (Bubendorf, Switzerland). SSR49059, SSR121463B, SSR126768A, and SSR149415 were kindly provided by Sanofi (Toulouse, France).
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