Primary mouse dermal fibroblasts were prepared from the dorsal skins of 8-week-old adult mice, as described previously.[8 ] Dermal fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) with medium changes every 2–3 days. Cells at passage 5 or less were harvested for subsequent experiments. Murine melan-a melanocytes were kindly provided by Dr. Dorothy C. Bennett (St. George's Hospital, London, UK).[9 (link)] Melan-a melanocytes were routinely cultured in RPMI1640 medium supplemented with 5% FBS, 100 mmol/L mercaptoethanol, 2 mmol/L L-glutamine, and 200 nmol/L phorbol-12-myristate acetate (PMA) (Sigma Aldrich, St. Louis, MO, USA).
Phorbol 12 myristate acetate pma
Manufactured by Merck Group
Sourced in United States
Phorbol-12-myristate acetate (PMA) is a chemical compound used as a laboratory reagent. It functions as a protein kinase C (PKC) activator, which is a key component in various cellular signaling pathways. PMA is commonly utilized in research studies to stimulate and investigate the effects of PKC activation on cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Thp 1 cell, by American Type Culture Collection (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Dimethyl itaconate, by Merck Group (1 mentions)
Sulforaphane, by Merck Group (1 mentions)
Fetal calf serum (fcs), by R&D Systems (1 mentions)
Lps from e coli 0111 b4, by InvivoGen (1 mentions)
Sendai virus cantrell strain, by Charles River Laboratories (1 mentions)
4 octyl itaconate, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Thp 1 monocytes, by American Type Culture Collection (1 mentions)
Magic redtm cathepsin b assay kit, by ImmunoChemistry Technologies (1 mentions)
Mts kit, by Promega (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Poly 1 c, by R&D Systems (1 mentions)
Thp 1 monocytes, by Merck Group (1 mentions)
10 protocols using phorbol 12 myristate acetate pma
1
Isolation and Culture of Primary Mouse Dermal Fibroblasts and Melanocytes
2
Modulation of THP-1 Macrophages by Stimuli
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THP-1 cells (ATCC) were cultured in RPMI 1640 (Thermo) supplemented with 10% FCS (R&D) and 1% Pen/Strep (Thermo). LUCAT1−/− THP-1 have been generated previously (10 (link)). Cells were differentiated into macrophage-like cells using 10 ng/mL phorbol-12-myristate acetate (PMA, Sigma) for 24 h, followed by media change and resting in PMA-free medium for another 24 h before stimulation. Cells were treated with 200 ng/mL LPS from E. coli 0111:B4 (Invivogen), Sendai virus Cantrell strain (Charles River Laboratories), 10 ng/mL IFN-α 2b (Gemini), 100 µM to 250 µM 4-octyl itaconate, 250 µM di-methyl itaconate, 5 µM sulforaphane (all Sigma), or 500 nM diABZI-4 (GSK) (19 (link)).
3
Evaluating Anti-Inflammatory Effects of SLNs
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In order to evaluate the cytotoxic and the anti-inflammatory effect of the CU- and RV-based SLNs (containing or not LNA), we treated two different cell types, the immortalized human NCTC 2544 keratinocytes and the THP-1 human monocytic leukemia cells, both cell types extensively used to mimic the cells normally present in the skin and both involved in the modulation of the inflammatory process underlying atopic dermatitis.
NCTC 2544 cells (a kind gift from Dr. R. De Bellis, Urbino, Italy) were maintained in Dulbecco’s modified minimal essential medium (DMEM) containing 2 mM glutamine, antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and 10% fetal bovine serum (FBS). THP-1 monocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in RPMI 1640 culture medium containing glutamine (2 mM) and 10%FBS. The differentiation of THP-1 cells into M2 macrophages was obtained by exposing the cells to 320 nm phorbol-12-myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) for 24 h.
NCTC 2544 cells (a kind gift from Dr. R. De Bellis, Urbino, Italy) were maintained in Dulbecco’s modified minimal essential medium (DMEM) containing 2 mM glutamine, antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and 10% fetal bovine serum (FBS). THP-1 monocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in RPMI 1640 culture medium containing glutamine (2 mM) and 10%FBS. The differentiation of THP-1 cells into M2 macrophages was obtained by exposing the cells to 320 nm phorbol-12-myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) for 24 h.
4
Synthesis and Characterization of Metal Oxide Nanoparticles
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MeONPs were in-house synthetized, donated, or purchased from commercial sources as outlined in Table S1; the in-house synthesis of (George et al. 2011 (link)) and ZnO (Tani et al. 2002 (link)) was conducted by a flame spray pyrolysis reactor as previously described; deionized (DI) water was obtained from a Milli-Q® water purification system (Millipore); THP-1 cells were purchased from ATCC; Corning® RPMI 1640 (CAT 10-041-CV) were purchased from Corning Inc.; 10% fetal bovine serum (CAT 100-500) was purchased from GeminiBio. Phorbol 12-myristate acetate (PMA) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. MTS kit was purchased from Promega. Magic cathepsin B assay kit was purchased from Immunochemistry Technologies; , IL-6, , and MCP-1 ELISA kits were purchased from BD Biosciences.
5
Differentiation of THP-1 Monocytes into Macrophages
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THP-1 cells were received from Vinay Nandicoori at the National Institute of Immunology (Delhi). THP-1 cells were passaged in complete RPMI medium [RPMI medium supplemented with 10% fetal bovine serum (FBS), HiMedia], penicillin (100 IU, Sigma-Aldrich), and streptomycin (100 mg/L, Sigma-Aldrich) and cultured at 37°C and 5% CO2. The THP-1 monocytes were differentiated to macrophages by treatment with 0.025 mg/L phorbol 12-myristate acetate (PMA, Sigma-Aldrich) for 24 h at 37°C and 5% CO2. Differentiated THP-1 cells were then cultured in fresh complete RPMI medium for 24 h prior to further treatment or infection.
6
Differentiation and Activation of THP-1 Macrophages
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In this experimental study, human THP-1 monocytes
were purchased from the American Type Culture
Collection (Manassas, VA, USA) and cultured in RPMI-
1640 medium that contained 10% heat-inactivated
foetal bovine serum (FBS, Gibco, USA) and 100 U/
mL penicillin-streptomycin. Cells were maintained
in an atmosphere of 5% CO2 at 37˚C. Exponential-phase
cells were used in the following assays.
THP-1 monocytes were induced to differentiate into
macrophages in vitro. Simply, THP-1 monocytes suspended
in RPMI-1640 medium were seeded in 6-well plates at a
density of 2×105 cells/mL. Then, 100 ng/mL phorbol-12-
myristate acetate (PMA) (Sigma, St. Louis, MO, USA) was
added to the THP-1 monocytes. After a 48-hour incubation
period, the adherent macrophages were used in the following
assays (THP1-Mφs). For polyI:C treatment, THP-1
monocytes were incubated with 100 ng/mL PMA for 6 hours,
and then treated with 25 μg/mL polyI:C (R&D Systems,
Minneapolis, MN, USA). After 42 hours of incubation, the
adherent macrophages were used in the following assays
(polyI:C-stimulated THP1-Mφs).
were purchased from the American Type Culture
Collection (Manassas, VA, USA) and cultured in RPMI-
1640 medium that contained 10% heat-inactivated
foetal bovine serum (FBS, Gibco, USA) and 100 U/
mL penicillin-streptomycin. Cells were maintained
in an atmosphere of 5% CO2 at 37˚C. Exponential-phase
cells were used in the following assays.
THP-1 monocytes were induced to differentiate into
macrophages in vitro. Simply, THP-1 monocytes suspended
in RPMI-1640 medium were seeded in 6-well plates at a
density of 2×105 cells/mL. Then, 100 ng/mL phorbol-12-
myristate acetate (PMA) (Sigma, St. Louis, MO, USA) was
added to the THP-1 monocytes. After a 48-hour incubation
period, the adherent macrophages were used in the following
assays (THP1-Mφs). For polyI:C treatment, THP-1
monocytes were incubated with 100 ng/mL PMA for 6 hours,
and then treated with 25 μg/mL polyI:C (R&D Systems,
Minneapolis, MN, USA). After 42 hours of incubation, the
adherent macrophages were used in the following assays
(polyI:C-stimulated THP1-Mφs).
7
Evaluation of Adjuvant Formulations in C57BL/6J Mice
8
Modulating Autophagy in THP-1 Macrophages
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The human monocytic leukaemia cell line THP-1 was obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Cells were cultured in RPMI 1640 medium (Gibco, USA) containing streptomycin (100 µg/mL), β-mercaptoethanol (0.05 mM) (Sigma-Aldrich, USA) and foetal bovine serum (10%) (Biological Industries, USA) at 37 °C with 5% CO2. THP-1 monocytes (1×106 cells/mL) were treated with phorbol-12-myristate acetate (PMA) (160 nM) (Sigma-Aldrich, USA) for 3 days to induce differentiation into macrophage-like cells.
The cells were cultured under different conditions. Cells that maintained in RPMI 1640 medium for 6 h were served as controls. Two groups of cells (low and high GV groups) were cultured in a low or high GV condition, as they were grown in routine RPMI 1640 medium and 5 mol/L D-glucose (low GV) or 25 mol/L D-glucose (high GV) alternately every 3 hours for 3 days. Another two groups of cells (high GV + rapamycin group and high GV + 3-methyladenine group) were pretreated with an autophagy inducer rapamycin (100 nmol/L; Selleck, USA) or an autophagy inhibitor 3-methyladenine (5 mmol/L; Sigma-Aldrich, USA) for 24 h, followed by cultured in the high CV condition mentioned above for 3 days. Western blot assay was utilized to quantified the expression levels of G3BP1 and NLRP3 in the above five groups.
The cells were cultured under different conditions. Cells that maintained in RPMI 1640 medium for 6 h were served as controls. Two groups of cells (low and high GV groups) were cultured in a low or high GV condition, as they were grown in routine RPMI 1640 medium and 5 mol/L D-glucose (low GV) or 25 mol/L D-glucose (high GV) alternately every 3 hours for 3 days. Another two groups of cells (high GV + rapamycin group and high GV + 3-methyladenine group) were pretreated with an autophagy inducer rapamycin (100 nmol/L; Selleck, USA) or an autophagy inhibitor 3-methyladenine (5 mmol/L; Sigma-Aldrich, USA) for 24 h, followed by cultured in the high CV condition mentioned above for 3 days. Western blot assay was utilized to quantified the expression levels of G3BP1 and NLRP3 in the above five groups.
9
Multicolor Flow Cytometry of T-cell Subsets
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APC-Cy7-labeled anti-CD3, PE-Cy7-labeled anti-IL-10, and isotype-matched control Igs were obtained from Biolegend (San Diego, CA, United States). PE-labeled anti IL-17A, and isotype-matched Ig control were obtained from eBioscience [San Diego, CA, United States]. FITC-labeled anti-CD4, PE-CF594-labeled anti-CD8 and isotype-matched control Ig were obtained from Becton Dickinson Horizon (San Jose, CA, United States). PerCP-labeled anti-LAP [TGF-β1] and isotype-matched control Ig were obtained from R&D Systems (Minneapolis, MN, United States). APC-labeled anti-Foxp3, the Foxp3 staining buffer set, and isotype-matched control Ig were obtained from eBioscience (San Diego, CA, United States). LIVE/DEAD ® Fixable Aqua Dead Cell Stain Kit was obtained from Life Technologies (Carlsbad, CA, United States). Phorbol-12-myristateacetate (PMA) and ionomycin were obtained from Sigma-Aldrich. FITC-labeled anti-CD4, isotype-matched control Igs, and Monensin solution (Golgi Stop) were obtained from BD Pharmingen (San Jose, CA, United States). For confocal microscopy imaging, polyclonal rabbit anti-CD4 (Novus, Colorado, United States), Donkey anti-rabbit-AlexaFluor-568 (Abcam, Cambridge, United Kingdom), monoclonal mouse anti-human LAP (R&D Systems) and goat anti-mouse AlexaFluor-647 (Abcam) were used.
10
Nanoparticle Synthesis: Protocols and Materials
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FeCl2·4H2O, FeCl3·6H2O and NOR used in the nanoparticle synthesis were purchased from Sigma-Aldrich, Germany. 25% ammonia solution was purchased from Merck, Germany (GR grade).
RPMI-1640 and fetal bovine serum (FBS) used for the culturing of THP1 cells were purchased from HiMedia, India. Phorbol 12 myristate acetate (PMA) used for THP1 cell differentiation was purchased from Sigma-Aldrich, Germany.
RPMI-1640 and fetal bovine serum (FBS) used for the culturing of THP1 cells were purchased from HiMedia, India. Phorbol 12 myristate acetate (PMA) used for THP1 cell differentiation was purchased from Sigma-Aldrich, Germany.
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