CD14+ monocytes (1 × 106 cells/well) were cultured in RPMI medium (Sigma Aldrich) containing 1% Penicillin/Streptomycin, 1% glutamine, 1% Fungizone and 10% FCS, supplemented with 50 ng/mL human recombinant M‐CSF (PeproTech) for 7 days. For use in β‐oxidation assays, M‐CSF‐differentiated macrophages were polarized with 100 ng/mL IL‐4 (Novartis) for 2 days in the presence of 10 ng/mL M‐CSF. The HDAC inhibitors SAHA (Cat.No.10009929, Cayman Chemicals) and MS‐275 (Cat.No.T6233, Target Mol) were added during the last 48 h. For analysis of pro‐inflammatory cytokine gene expression, M‐CSF‐differentiated macrophages were stimulated with 100 ng/mL LPS (E. coli 055:B5, Cat.no. L4005, Sigma) and treated with DMSO, MS‐275, or SAHA in concentrations as indicated for 24 h. For detachment, adherent macrophages were washed with PBS and incubated with 300 µL Gibco™TrypLE™Select(10×) (Gibco, Life Technologies) for 15 min at 37°C, and collected with a cell scraper after adding 300 µL PBS.
Gibco tryple select 10
Manufactured by Thermo Fisher Scientific
Gibco™TrypLE™Select(10×) is a cell dissociation reagent used for the detachment of adherent cells from cell culture vessels. It is a concentrated, recombinant trypsin-like protease that can be diluted and used as a replacement for traditional trypsin-EDTA solutions.
Automatically generated - may contain errors
Lab products found in correlation
M csf, by Novartis (2 mentions)
Rpmi medium, by Merck Group (2 mentions)
Interleukin 4 (il 4), by Novartis (2 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (1 mentions)
Ms 275, by Cayman Chemical (1 mentions)
Recombinant human gm csf, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
M csf, by Merck Group (1 mentions)
Lps e coli 055 b5, by Merck Group (1 mentions)
Vorinostat, by Cayman Chemical (1 mentions)
2 protocols using gibco tryple select 10
1
Macrophage Polarization and Cytokine Expression
2
Macrophage Differentiation and Polarization Protocol
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For macrophage differentiation and polarization, CD14+ monocytes (1 × 106 cells/well) were isolated and characterized by flow cytometry as described previously15 , 16 and seeded in RPMI medium (Sigma‐Aldrich) containing 1% Penicillin/Streptomycin, 1% glutamine, 1% Fungizone, and 10% fetal calf serum, supplemented with either 50 ng/mL human recombinant GM‐CSF or M‐CSF (PeproTech) for 7 days. For β‐oxidation assays, M‐CSF‐differentiated macrophages were polarized into an anti‐inflammatory phenotype with 100 ng/ml IL‐4 (Novartis) and 10 ng/ml M‐CSF for 2 days. To analyse pro‐inflammatory cytokine gene expression, M‐CSF‐differentiated macrophages were stimulated with 100 ng/ml LPS (E. coli 055:B5, #L4005, Sigma) and treated with vehicle control or Vorinostat (#10009929, Cayman Chemicals) for 24 h. ELISA to measure secreted IL12p40 was described previously.16 To determine mean cell size and number of adherent or nonadherent cells, a CASY automated cell counter (Omni Life Sciences) was used. For detachment, adherent macrophages were incubated with 300 µL Gibco™ TrypLE™ Select (10×) (Gibco, Life Technologies) for 15 min at 37°C shaking every few minutes and was gently removed with a cell scraper.
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