Mature (6 d) monocyte-derived MΦs were prepared in 3 replicates using 25 cm2 tissue culture flasks (Falcon/Corning) containing RPMI 1640, w/L-glutamine (Lonza) media, supplemented with 1 mM of sodium pyruvate (Lonza), 1X MEM NEAAs (Gibco), 10% (v/v) FBS (ATCC), and 50 ng/mL rHU-MCSF (PeproTech). Three different MΦ activation groups were assessed and compared, including M0, M1, and M2a MΦs. Cell cultures corresponding to each MΦ activation state, and parallel sham media controls, corresponding to macrophage activation and cell culture media added to 25 cm2 tissue culture flasks that did not contain mature monocyte-derived MΦs, were set up and incubated for 72 hrs at 37 °C, 5% CO2. MΦ activation stimuli included 100 ng/mL of LPS and 50 ng/mL of recombinant human IFN-γ (rHU-IFNγ) for M1 MΦs (Sigma Aldrich and PeproTech, respectively), whereas no additional stimuli were added to M0 MΦs. Then, 20 ng/mL recombinant human IL-4 (rHU-IL4; PeproTech) was utilized to generate M2a MΦs. Following culture and activation, the phenotypic characterization of primary human monocyte-derived MΦs (MoMΦs) was conducted using flow cytometry (Figure S8 ).
Rhu mcsf
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RHU-MCSF is a laboratory instrument designed to measure the concentration of macrophage colony-stimulating factor (M-CSF) in biological samples. It utilizes a recombinant human M-CSF (RHU-MCSF) as the standard for quantification. The core function of this product is to provide accurate and reliable M-CSF measurement capabilities for researchers and scientists in various fields of study.
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Lab products found in correlation
Sodium pyruvate, by Lonza (2 mentions)
Rhu il 4, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Tissue culture flask, by Corning (1 mentions)
Tissue culture flasks, by BD (1 mentions)
Mem non essential amino acids neaas, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
3 protocols using rhu mcsf
1
Characterization of Human Monocyte-Derived Macrophages
2
Differentiation of Primary Human Monocyte-derived Macrophages
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To generate primary human monocyte-derived MΦs, CD14+ monocytes were cultured in 25 cm2 tissue culture flasks (1 × 106 cells per mL; Falcon/Corning) in Roswell Park Memorial Institute (RPMI) medium -1640, w/L-glutamine (Lonza, Bend, OR, USA) media, supplemented with 1 mM of sodium pyruvate (Lonza), 1X MEM non-essential amino acids (NEAAs; Gibco, ThermoFisher Scientific, USA), 10% (v/v) fetal bovine serum (FBS; ATCC, Manassas, VA, USA), and 50 ng/mL recombinant human M-CSF (rHU-MCSF; PeproTech, Rocky Hill, NJ, USA) for 6 d. Media and cytokines were replenished every 3 d.
3
Macrophage Polarization and Mycobacterial DNA Stimulation
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We utilized human monocytic cells (THP-1) under an established protocol for macrophage induction and polarization, creating M1, M1 activated (M1a), and M2 macrophages (Rey–Giraud et al., 2012 (link)). 5 × 105 cells/ml in 12-well tissue culture treated plates for 6 days in the presence of either 100 ng/ml rHuGM-CSF (M1) or 100 ng/ml rHuM-CSF (M2). For M1 activation, monocytes were first incubated with rHuM-CSF (Peprotech) for 3 days followed by stimulation with 10 ng/ml LPS (Sigma) and 50 ng/ml rHuIFN-γ (Roche) for 3 additional days (Rey–Giraud et al., 2012 (link)). Stimulation was performed with 500 ng of genomic DNA from three different strains of M. leprae, NHDP (NT-19350), Br4923 (NR-19351), and Thai-53 (NR-19352), acquired from BEI Resources (Manassa, VA). We utilized these strains as each one of them belongs to a different genetic subtype (Singh and Cole, 2011 (link), Truman et al., 2011 ).
Genomic DNA was isolated from contaminating proteins and polysaccharides by organic extraction and precipitation with isopropanol respectively (Belisle and Sonnenberg, 1998 (link)). Polyethilenimine (PEI) (Polyplus) was used as an endo-lysosomal bacterial nucleic acid delivery system (Cervantes et al., 2013 (link), Suh et al., 2012 (link), Bieber et al., 2002 (link)), according to manufacturer's instructions.
Genomic DNA was isolated from contaminating proteins and polysaccharides by organic extraction and precipitation with isopropanol respectively (Belisle and Sonnenberg, 1998 (link)). Polyethilenimine (PEI) (Polyplus) was used as an endo-lysosomal bacterial nucleic acid delivery system (Cervantes et al., 2013 (link), Suh et al., 2012 (link), Bieber et al., 2002 (link)), according to manufacturer's instructions.
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