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Phorbol myristate acetate (pma)

Manufactured by Cayman Chemical
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PMA is a laboratory reagent used in chemical analysis and research. It is a white crystalline solid that functions as a chemical standard or reference material. PMA is primarily employed in analytical procedures to aid in the identification and quantification of specific compounds.

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Lab products found in correlation

Ionomycin, by Cayman Chemical (3 mentions) Lipopolysaccharides (lps), by Merck Group (3 mentions) Macsxpress neutrophil extraction kit, by Miltenyi Biotec (3 mentions) Macsxpress separator, by Miltenyi Biotec (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (3 mentions) Rotenone, by Abcam (2 mentions) Cell tak, by Corning (2 mentions) Antimycin a, by Abcam (2 mentions) Xf96e extracellular flux analyzer, by Agilent Technologies (2 mentions) Duoset elisa development system, by R&D Systems (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Ifn γ, by Thermo Fisher Scientific (2 mentions) Gentamicin, by Thermo Fisher Scientific (2 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Progesterone, by Merck Group (2 mentions) Estradiol, by Merck Group (2 mentions) Turbo dna free 50 reactions, by Thermo Fisher Scientific (2 mentions) 8 bromoadenosine 3 5 cyclic monophosphate 8 br camp pcr primers, by Merck Group (2 mentions) Ultrapure agarose, by Thermo Fisher Scientific (2 mentions) Ultrapure glycogen, by Thermo Fisher Scientific (2 mentions)

29 protocols using phorbol myristate acetate (pma)

1

Intracellular Staining of T Cells

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For intracellular staining, T cells were restimulated with 5 ng/ml PMA (Cayman Chemical) and 200 ng/ml ionomycin (Cayman Chemical) for 4 h in the presence of GolgiPlug (BD Pharmingen). Subsequently, surface staining was performed at 4°C for 30 min. Cells were fixed and permeabilized using Cytofix/Cytoperm plus Fixation/Permeabilization Kit (BD Pharmingen). For analysis of intranuclear markers, cells were fixed and stained using the FoxP3 intranuclear staining kit (eBioscience), again incubating cells at 4°C for 30 min both during fixation and intranuclear staining. Antibodies are summarized in Table 1. Analysis was performed using a Gallios Flow Cytometer (Beckman Coulter) and results were analyzed with FlowJo software (Tree Star).
Herold M., Breuer J., Hucke S., Knolle P., Schwab N., Wiendl H, & Klotz L. (2017). Liver X receptor activation promotes differentiation of regulatory T cells. PLoS ONE, 12(9), e0184985.
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2

In Vitro Macrophage Polarization

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Human monocytic THP-1 cells(ATCC TIB-202) were acquired from S. Minucci (IEO Milan, Italy) and maintained in Roswell Park Memorial Institute medium(RPMI 1640, Thermofisher) culture medium supplemented with 10% fetal bovine serum(Gibco, 10270106), 1mM pyruvate(Life Technologies), 2mM GlutaMAX(Thermofisher, 35050-038). THP-1 monocytes are differentiated into macrophages by 48h incubation with 150nM phorbol 12-myristate 13-acetate(PMA, Cayman Chemicals; Cay10008014) followed by 24h incubation in RPMI medium. Macrophages were polarized into M1 macrophages by incubation with 20ng/ml of IFNγ(R&D system, 285-IF) and 10pg/ml of LPS(Sigma, L2630). Macrophage M2 polarization was obtained by incubation with 20ng/ml of interleukin 4(Sigma, A3134) and 20ng/ml of interleukin 13(PeproTech, 200-13).
Schmitt M.J., Dramaretska Y., Barozzi I., Göhrig A., Kertalli S., Großmann M., Naumann H., Sanchez-Bailon M.P., Hulsman D., Glass R., Squatrito M., Serresi M, & Gargiulo G. (2020). Phenotypic mapping of pathological crosstalk between glioblastoma and innate immune cells by synthetic genetic tracing. Cancer discovery, 11(3), 754-777.
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3

Oxidative Burst Evaluation in Mice

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Mice were primed with 3D 6-Acyl PHAD at 48 and 24 hours prior to euthanasia, followed by peritoneal lavage with 5 mL of PBS. Peritoneal leukocytes were prepared for flow cytometry as described above. Cells were resuspended at 1 × 106 cells/mL, incubated with dihydrorodamine 123 for 15 minutes, followed by incubation with phorbol myristate acetate (PMA, 200 nM) for 45 minutes to provoke generation of reactive oxygen species, according to manufacturer’s recommendations (Cayman Chemical, Ann Arbor, MI). After stimulation, dihydrorhodamine 123 MFI was measured by flow cytometry. Forward versus side scatter was used to identify neutrophils, and monocytes.
Hernandez A., Luan L., Stothers C.L., Patil N.K., Fults J.B., Fensterheim B.A., Guo Y., Wang J., Sherwood E.R, & Bohannon J.K. (2019). Phosphorylated Hexaacyl Disaccharides Augment Host Resistance Against Common Nosocomial Pathogens. Critical care medicine, 47(11), e930-e938.
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4

Neutrophil Oxidative Burst Kinetics

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Suspended neutrophils were plated in culture wells pre-coated with Cell-Tak™ (Corning), spun at the 200 x g for 1 min. with minimal acceleration/deceleration, and then incubated for 1h at 37 °C. Murine neutrophils were plated at 2 × 105 cells/ well, in RPMI 1640 media without sodium bicarbonate. Human neutrophils were plated at 5 × 104 cells/ well, in RPMI 1640 media supplemented with 0.1% human serum albumin. Inhibitor (G6PDi-1, 50µM) or vehicle control were added just prior to starting assay. Mouse neutrophils were also treated with rotenone (0.5µM, BioVision) + antimycin A (0.5 µM, BioVision) at t = 20 min. Oxidative burst was stimulated with PMA (100 nM, Cayman Chemical); oxygen consumption rate using the XF-96e extracellular flux analyzer (Seahorse Bioscience).
Ghergurovich J.M., García-Cañaveras J.C., Wang J., Schmidt E., Zhang Z., TeSlaa T., Patel H., Chen L., Britt E.C., Piqueras-Nebot M., Gomez-Cabrera M.C., Lahoz A., Fan J., Beier U.H., Kim H, & Rabinowitz J.D. (2020). A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nature chemical biology, 16(7), 731-739.
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5

Neutrophil Oxidative Burst Kinetics

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Suspended neutrophils were plated in culture wells pre-coated with Cell-Tak™ (Corning), spun at the 200 x g for 1 min. with minimal acceleration/deceleration, and then incubated for 1h at 37 °C. Murine neutrophils were plated at 2 × 105 cells/ well, in RPMI 1640 media without sodium bicarbonate. Human neutrophils were plated at 5 × 104 cells/ well, in RPMI 1640 media supplemented with 0.1% human serum albumin. Inhibitor (G6PDi-1, 50µM) or vehicle control were added just prior to starting assay. Mouse neutrophils were also treated with rotenone (0.5µM, BioVision) + antimycin A (0.5 µM, BioVision) at t = 20 min. Oxidative burst was stimulated with PMA (100 nM, Cayman Chemical); oxygen consumption rate using the XF-96e extracellular flux analyzer (Seahorse Bioscience).
Ghergurovich J.M., García-Cañaveras J.C., Wang J., Schmidt E., Zhang Z., TeSlaa T., Patel H., Chen L., Britt E.C., Piqueras-Nebot M., Gomez-Cabrera M.C., Lahoz A., Fan J., Beier U.H., Kim H, & Rabinowitz J.D. (2020). A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nature chemical biology, 16(7), 731-739.
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6

Neutrophil ROS Production Assays

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Cytochrome C reduction assays or Neutrophil/monocyte respiratory burst assay kits (Cayman chemical) were used. Freshly isolated CD66b+ LDNs and/or NDNs were washed and resuspended at 2x106 (link) cells/ml in Hank’s Balanced Salt Solution (HBSS) pH 7.4, supplemented with 10 % FBS, 0.5 mM CaCl2 and 1 mg/ml glucose. O2- production in response to 20 ng/ml PMA (Sigma) was assessed by the Cytochrome C reduction assay (Cayman), as previously described51 (link). For flow cytometry analysis of ROS, 1x105 (link) mononuclear cells or granulocytes were incubated with Dihydrorhodamine-123 (Cayman chemical) and left untreated or stimulated with PMA 20 ng/ml for 15 or 30 minutes. Cells were stained with fluorochrome-conjugated antibodies as described above, acquired at FACSCanto II using DIVA software v8.0.2 (BD Biosciences) and analyzed with FlowJo v10.6.2 (TreeStar). LDNs and NDNs were identified after gating on Lin- CD15+ cells in the PBMC and granulocyte fraction, respectively. A detailed reagent list is reported in Supplementary Table 44.
Montaldo E., Lusito E., Bianchessi V., Caronni N., Scala S., Basso-Ricci L., Cantaffa C., Masserdotti A., Barilaro M., Barresi S., Genua M., Vittoria F.M., Barbiera G., Lazarevic D., Messina C., Xue E., Marktel S., Tresoldi C., Milani R., Ronchi P., Gattillo S., Santoleri L., Di Micco R., Ditadi A., Belfiori G., Aleotti F., Naldini M.M., Gentner B., Gardiman E., Tamassia N., Cassatella M.A., Hidalgo A., Kwok I., Ng L.G., Crippa S., Falconi M., Pettinella F., Scapini P., Naldini L., Ciceri F., Aiuti A, & Ostuni R. (2022). Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress. Nature immunology, 23(10), 1470-1483.
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7

Murine Lung Epithelial Cell Culture

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Murine lung epithelial (MLE12) cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured with HITES medium containing 10% FBS at 37°C in 5% CO2 incubator. V5 tag antibody, mammalian expressional plasmid pcDNA3.1/His-V5 TOPO, and Escherichia coli Top 10 competent cells were from Invitrogen (Carlsbad, CA, USA). A549 (human lung carcinoma cell line) Beas2B (human lung bronchial epithelial cell line), HBE (human bronchial epithelial cells) and HEK293T (human embryonic kidney) were from ATCC. HECW1 siRNA, Leupeptin, and β-actin antibody were from Sigma Aldrich (St. Louis, MO, USA). MG-132 was from Calbiochem (La Jolla, CA, USA). Immunobilized protein A/G beads, ubiquitin, HA tag, TTF1, and control IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HECW1 antibody was from Sabbiotech (College Park, MD, USA). GAPDH antibody was from Proteintech (Rosemont, IL, USA). PMA was from Cayman Chemical Company (Ann Arbor, MI, USA). All materials in highest grades uses in the experiments are commercially available.
Liu J., Dong S., Li L., Wang H., Zhao J, & Zhao Y. (2019). The E3 ubiquitin ligase HECW1 targets thyroid transcription factor 1 (TTF1/NKX2.1) for its degradation in the ubiquitin-proteasome system. Cellular signalling, 58, 91-98.
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8

Quantifying Immune Responses to Anti-PD-L1 and Anti-CTLA-4 Therapies

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Example 10

Vero-αHis cells were seeded in 15 cm cell culture dishes and infected with MeVac encoding anti-PD-L1, anti-CTLA-4, CD80-Fc or IgG1-Fc with M01=0.03. Supernatants were collected (15 ml per plate) when syncytia had spread over the whole cell layer (ca. 36 h post infection). 2×105 MC38cea cells were incubated with 2 ml medium collected from the Vero-αHis infected with the respective viruses for 5 min with rotation at room t° and pelleted by centrifugation 5 min at 300×g. The procedure was repeated six times. The treated cells were resuspended in 100 μl activation medium—RPMI 1640 supplemented with 5% FCS, 1% Penicillin-Streptomycin (Life Technologies), 500 μM ionomycin (Cayman Chemical Company, Hamburg, Germany) and 5 μM PMA (Cayman Chemical Company) and seeded in 96-well plate. 2×105 freshly isolated splenocytes from C57BL/6J mouse in 100 μl activation medium were added per each well with the treated MC38cea cells. Cells were cocultivated 24 h at 37° C. 5% CO2 and supernatants collected subsequently. IFN-γ concentration was determined using mouse IFN gamma ELISA Ready-SET-Go!® (eBioscience, Frankfurt am Main, Germany) according to the instructions of the manufacturer.

US10344067B2. RNA viruses expressing IL-12 for immunovirotherapy (2019-07-09). DEUTSCHES KREBSFORSCHUNGSZENTRUM [DE], RUPRECHT-KARLS UNIVERSITAET [DE]. Inventors: Guy Ungerechts [DE], Christine Engeland [DE], Ruta Veinalde [DE].
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9

Isolation and Stimulation of Neutrophils

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PMNs were isolated from 100 μl whole blood by magnetic bead-based negative selection using the MACSxpress Neutrophil Extraction Kit (Miltenyi Biotec) and a strong magnet (MACSxpress Separator, Miltenyi Biotec). Harvested PMNs were resuspended in PBS (with Ca2+ and Mg2+) by centrifugation at 1,000 r.p.m. for 5 min. PMNs were stimulated with PMA at 20 nM (Cayman Chemical Co. Inc.) or vehicle (ethanol v/v 0.02%) and incubated for 30 min at 37 °C in 5% CO2. After incubation, PMNs were resuspended in PBS buffer by a single centrifugation at 1,000 r.p.m. for 5 min.
Jundi B., Ryu H., Lee D.H., Abdulnour R.E., Engstrom B.D., Duvall M.G., Higuera A., Pinilla-Vera M., Benson M.E., Lee J., Krishnamoorthy N., Baron R.M., Han J., Voldman J, & Levy B.D. (2019). Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity. Nature Biomedical Engineering, 3(12), 961-973.
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10

Neutrophil Degranulation Assay

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Neutrophil degranulation, based on the release of elastase into culture supernatants, was assessed using another DuoSet ELISA Development System (R&D Systems, Inc., Minneapolis, MN). In brief, cells were plated in 24-well dishes and grown until reaching 80% confluency; at that point, the medium was removed and the cells were then exposed to 50 μg AgNP/ml medium for 24 h. After this period, the cells were washed with fresh media and the media containing 1 μg/ml phorbol myristate acetate (PMA, Cayman) or medium only (for basal activity measurements) was then added to the cells. After 1 h, culture well supernatants were collected and neutrophil elastase was then quantified in the same manner as described in the section “Quantification of cytokine release by ELISA”.
Alsaleh N.B., Minarchick V.C., Mendoza R.P., Sharma B., Podila R, & Brown J.M. (2019). Silver nanoparticle immunomodulatory potential in absence of direct cytotoxicity in RAW 264.7 macrophages and MPRO 2.1 neutrophils. Journal of immunotoxicology, 16(1), 63-73.
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