The human monocytic leukaemia cell line THP-1 (ECACC) was used routinely between passage 8 and 24 and maintained in complete growth RPMI 1640 medium (Lonza) supplemented with 10% (v/v) FBS (Sera lab), 2 mM L -glutamine, 100 unit/ml penicillin and 100 μg/ml streptomycin (P/S) (Lonza). Cells were kept at 37°C, under 5% CO2 in a humidified incubator.
For THP-1 differentiation into macrophage-like subsets, the cells were plated (at density of 1×106 cell/ml) in complete medium and treated with PMA (M1-like; 25 ng/ml; 3 days followed by 5 days resting in fresh medium) or 1,25-(OH)2-Vitamin D3 (Sigma–Aldrich; M2-like; 10 nM; 7 days) [12 (link)].
Cells were stimulated with 100 ng/ml LPS derived from Escherichia coli K12-LPS (Autogen-Bioclear Ltd.) for the indicated time intervals. Dead cells were aspirated and the live cells and supernatant were harvested by centrifugation at 200g for 5 min, and stored at −20°C until the day of analysis. For cell counting, adherent cells (M1-like) were de-attached by trypsinization using 0.5% (v/v) of trypsin with 0.2% (v/v) EDTA (Sigma–Aldrich).
For THP-1 differentiation into macrophage-like subsets, the cells were plated (at density of 1×106 cell/ml) in complete medium and treated with PMA (M1-like; 25 ng/ml; 3 days followed by 5 days resting in fresh medium) or 1,25-(OH)2-Vitamin D3 (Sigma–Aldrich; M2-like; 10 nM; 7 days) [12 (link)].
Cells were stimulated with 100 ng/ml LPS derived from Escherichia coli K12-LPS (Autogen-Bioclear Ltd.) for the indicated time intervals. Dead cells were aspirated and the live cells and supernatant were harvested by centrifugation at 200
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