Polylactic acid (Mw = 20.2 kDa, Mn = 12.4kDa) was obtained from Lactel. H2N-PEG(5000)-OCH3 was obtained from Laysan. Anhydrous dimethylformamide, dichloromethane, diisopropylcarboimide, dimethylaminopyridine, potassium methoxide, camptothecin, polyvinyl alcohol, paraformaldehyde, Tween 80, and 1,1,1-trihydroxymethyl propane were obtained from the Sigma-Aldrich. Anhydrous dry ether, methanol, acetonitrile and dimethylsulfoxide were obtained from J.T. Baker. The 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine,4 Chlorobenzenesulfonate Salt (DiD) and DAPI stain were ordered from Invitrogen. Super frost microscope slides were obtained from Thermo Scientific. Donkey normal serum and Rabbit-anti-CD31 antibody was ordered from Abcam and the Donkey-anti-rabbit secondary antibody tagged with Alexa488 fluorophore was from Invitrogen. Cell titer blue was obtained from Promega. Microdialysis tubing was from Thermo Scientific. Phorbol 12-myristate 13-acetate (PMA) was from Abcam.
Phorbol 12 myristate 13 acetate
Manufactured by Abcam
Sourced in United Kingdom
Phorbol 12-myristate 13-acetate is a chemical compound that acts as a protein kinase C activator. It is commonly used as a laboratory tool in cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Anhydrous dimethylformamide, by Merck Group (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Dimethylaminopyridine, by Merck Group (1 mentions)
Superfrost microscope slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Dapi stain, by Thermo Fisher Scientific (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindodicarbocyanine, by Thermo Fisher Scientific (1 mentions)
4 chlorobenzenesulfonate salt did, by Thermo Fisher Scientific (1 mentions)
Potassium methoxide, by Merck Group (1 mentions)
Celltiter blue, by Promega (1 mentions)
2 aminopurine 2 ap, by Merck Group (1 mentions)
Luteolin, by Cayman Chemical (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
6 protocols using phorbol 12 myristate 13 acetate
1
Polylactic Acid Nanoparticle Synthesis and Characterization
2
Mammary Fibroblasts-Macrophage Co-culture
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Mammary fibroblasts were seeded into the base of a 6-well plate at 5 × 104 cells/mL in supplemented DMEM media, and grown to 90% confluence before serum starvation overnight. Separately, 2 mL of THP-1 monocyte cell suspension was added to the internal compartment of 24 mm trans-well with 0.4µm pore polycarbonate membrane (Corning, Corning, NY, USA; Cat#3412) at 0.5 × 106 cells/mL and were activated by 5 ng/mL of phorbol-12-myristate-13-acetate (Abcam, Cambridge, UK; Cat#ab120297) in supplemented RPMI media for 72 h to differentiate THP-1 cells to adherent M0 macrophages. Following removal of PMA, trans-well inserts were then placed into the wells containing mammary fibroblasts (0.5 × 106 cells/mL) and were incubated in supplemented, serum free DMEM. Co-culture studies were compared to macrophage-only or fibroblast-only controls, where the cell type was cultured with base medium only.
3
Polarization of THP-1 Macrophages
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THP-1 wt and THP-1 RTN1A+ cells were seeded on cover slips in a 24-well plate (2.5×104 cells/well) and polarized toward Mφs for 72 hr with 50 ng/ml phorbol 12-myristate 13-acetate (Abcam) in RPMI medium and supplements as described previously (Pinto et al., 2021 (link)).
4
Screening of Antimicrobial Compounds
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Luteolin was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). PKR inhibitors (2-aminopurine: 2-AP and C16) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Phorbol-12-myristate-13-acetate (PMA) was purchased from Abcam (Cambridge, UK). Guaiacol-parachlorophenol (Methocol®), which is composed of 30% guaiacol and 70% parachlorophenol, was obtained from Neo Dental Chemical Products Co., Ltd. (Tokyo, Japan). Lipopolysaccharides (LPS) from Escherichia coli 055:B5 (E. coli LPS) and Porphyromonas gingivalis (P. g LPS) were purchased from Sigma–Aldrich.
5
Generating M2-like Macrophages from THP-1 Cells
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THP‐1 cells were first treated with 100 ng/mL phorbol 12‐myristate 13‐acetate (Abcam) for 24 h to generate unstimulated macrophages, followed by incubation with IL‐4 (50 ng/mL, PeproTech) and IL‐13 (20 ng/mL, PeproTech) for 48 h for differentiation into M2‐like macrophages.
6
Myofibroblast-Macrophage Crosstalk in Co-culture
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Human foreskin fibroblasts (HFF-1) were cultured in Dulbecco modified Eagle medium without fetal bovine serum (FBS; Gibco, USA) for 12 hours, and then the culture medium was replaced by high glucose Dulbecco modified Eagle medium with 10% FBS. Fibroblast differentiation into myofibroblast was induced with TGF-β1(10 ng/mL) for 48 hours. Human myeloid leukemia mononuclear cells (THP-1) were cultured in 1640 Medium containing 10% FBS, differentiated into macrophages by a 48-hour incubation with 50 ng/mL phorbol 12-myristate 13-acetate (Abcam, Shanghai, China). Macrophages were polarized to M2 macrophages by adding 20 ng per mL IL-4 and IL-13 to the media for an additional 48 hours.
The Transwell co-culture assay was performed using the six-well transwell plates (Labselect, Shanghai, China). Myofibroblasts were applied to the lower compartment, whereas M2 macrophages were added to the upper compartment of the plates.
The Transwell co-culture assay was performed using the six-well transwell plates (Labselect, Shanghai, China). Myofibroblasts were applied to the lower compartment, whereas M2 macrophages were added to the upper compartment of the plates.
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