All chemicals, reagents, and kits were purchased at cell culture/molecular biology grade. Plasticware was invariably purchased, sterilized, disposed of, and treated for cell culture. Fetal bovine serum (FBS), Dulbecco’s phosphate buffer saline (D-PBS), Eagle’s minimum essential medium (MEM), penicillin/streptomycin solutions, trypsin, L-glutamine and non-essential amino acids were purchased from Corning-Fisher Scientific (Rodano, Italy). The compound 4′,6-Diamidino-2-Phenylindole (DAPI; Cat.: 28718-90-3), Triton X-100, phorbol 12-myristate 12-acetate (PMA; Cat. 16561-29-8), paraformaldehyde (PFA; Cat. 30525-89-4), bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Milano, Italy). Phalloidin TRITC-conjugated (Cat.: R415) and L-carnosine (Cat. 305-84-0) were purchased from Thermo Fisher Scientific (Monza, Italy).
Phorbol 12 myristate 12 acetate pma
Manufactured by Merck Group
Sourced in United States
Phorbol 12-myristate 13-acetate (PMA) is a chemical compound commonly used as a research tool in cell biology and biochemistry. It functions as a potent activator of protein kinase C, a family of enzymes involved in various cellular processes. PMA is widely used in laboratory settings to study signal transduction pathways, cell proliferation, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
D35c4 20 1.5 n, by Cellvis (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Thp 1, by American Type Culture Collection (2 mentions)
Raw264, by American Type Culture Collection (2 mentions)
Amaxa nucleofector kit, by Lonza (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
L glutamine, by Corning (1 mentions)
Trypsin, by Corning (1 mentions)
Non essential amino acids (neaa), by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Mpc 11, by BioLegend (1 mentions)
Percoll gradient centrifugation, by GE Healthcare (1 mentions)
Complete rpmi, by Merck Group (1 mentions)
5 protocols using phorbol 12 myristate 12 acetate pma
1
Cell Culture and Immunofluorescence Protocol
2
THP-1 Macrophage Differentiation and Nanoparticle Exposure
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THP-1 cells were purchased from ATCC (Manassas, VA) and cultured according to a standard protocol used by the NIEHS Nano GO Consortium [27] (link). Cells were cultured in suspension with RPMI-1640 Medium (Invitrogen, Carlsbad CA) containing 10% fetal bovine serum (Gibco) at 37°C and 5% CO2. Once confluent, the THP-1 cells were differentiated into a macrophage-like cell using 150 nM of 1α, 25-Dihydroxy-Vitamin D3 (EMD Millipore, Billerica MA). After the cells become semi-adherent, 10 ng/ml of lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis MO) was added to induce intracellular pro-IL-1β as well as 5 nM of phorbol 12-myristate 12-acetate (PMA) (Sigma-Aldrich) to further differentiate the monocytes to macrophages. Prior to nanoparticle exposure, 1×105 cells were seeded into 96-well plates (Costar, Corning NY). THP-1 cells were then exposed to increasing doses (5, 10, 50 & 100 µg/ml) of uncoated and Al2O3 coated MWCNTs (unsonicated vs. sonicated) as well as carbon black & Al2O3 nanoparticles (negative & positive control) for 24 hours. Cells were given a dose that was determined by the estimated number of nanotubes. Cell culture conditioned medium were collected at 24 hr for ELISA. MTS cytotoxicity assays were performed on the monolayer of cells (Figs S1 -S3
3
Live-cell Microscopy of Transfected Cell Lines
4
Isolation and Stimulation of Murine Immune Cells
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Single cell suspensions obtained from homogenized murine kidneys, livers and spleen were subjected to 44% (v/v) Percoll gradient centrifugation (GE Healthcare Life Sciences, Little Chalfont, UK) and washed twice with ice-cold PBS before stimulation. Cell suspensions from urinary bladders and lungs were not purified by Percoll gradient because of limited leukocyte yield.
5 × 105 cells were re-suspended in 500 μL of complete RPMI (Sigma-Aldrich, Gillingham, UK) with 50 ng/mL Phorbol 12-myristate 12-acetate (PMA) (Sigma-Aldrich, Gillingham, UK), 1 ug/mL ionomycin (Sigma-Aldrich, Gillingham, UK) and BD GolgiStopTM (BD Biosciences). The suspensions were vortexed quickly and incubated for 4 hours at 37 °C with 5% CO2. At the end of the stimulation, cells were washed with ice-cold PBS once and stained for surface antigens as described above. Following intracellular staining for IL-10 was performed using BD Cytofix/CytopermTM kit (BD Biosciences) and anti-mouse IL-10 antibody at dilution 1:100 (JES5-16E3; BD Pharmigen), or isotype control (MPC-11; Biolegend).
5 × 105 cells were re-suspended in 500 μL of complete RPMI (Sigma-Aldrich, Gillingham, UK) with 50 ng/mL Phorbol 12-myristate 12-acetate (PMA) (Sigma-Aldrich, Gillingham, UK), 1 ug/mL ionomycin (Sigma-Aldrich, Gillingham, UK) and BD GolgiStopTM (BD Biosciences). The suspensions were vortexed quickly and incubated for 4 hours at 37 °C with 5% CO2. At the end of the stimulation, cells were washed with ice-cold PBS once and stained for surface antigens as described above. Following intracellular staining for IL-10 was performed using BD Cytofix/CytopermTM kit (BD Biosciences) and anti-mouse IL-10 antibody at dilution 1:100 (JES5-16E3; BD Pharmigen), or isotype control (MPC-11; Biolegend).
5
Cell Culture and Transfection Techniques
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HeLa, HEK293T, THP-1, and RAW264.7 cells were obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's media (DMEM, Sigma-Aldrich, St Louis, MO, USA) or RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) under 37 °C at a 5% CO2 atmosphere. Phorbol 12-myristate 12-acetate (PMA) from Sigma Aldrich (St Louis, MO, USA) was used at 20 ng/mL to induce THP-1 differentiation to macrophage. DNA transfections were carried out by using the Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) or using an Amaxa nucleofector kit (Lonza) for transfection of plasmids into THP-1 and RAW264.7 cells. For live-cell imaging, cells were seeded in four-chamber 35mm glass-bottom dishes (D35C4-20-1.5-N, Cellvis, Mountain View, CA, USA) at 40-60% confluency, and imaged 24 hours after transfection.
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