THP-1 macrophage polarization
Description
Polarization of THP-1 -derived macrophages on aligned PCL scaffolds and in TCP monolayers
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Steps to reproduce
Frozen THP-1 cells were purchased from Sigma, thawed in 37° water bath, resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 0.85 g/l sodium bicarbonate, 1% Pen/Strep and 10% fetal bovine serum (FBS) and seeded at a seeding density of 3 x 105 cells per ml. Upon 80% confluency (8 x 105 cells/ml), cells were passaged and seeded in new T75 flasks at a seeding density of 3 x 105 cells per ml. THP-1 cells at passages 24-28 were used for subsequent experiments. Cells were seeded in 24-well plates. For priming THP-1 cells into macrophage-like cells, THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) at 5[2] and 20 ng/ml[3, 4] for 24 hours, followed by a 48h rest period in PMA free RPMI medium[2, 3]. THP-1 derived macrophages were incubated in RPMI medium supplemented with lipopolysaccharide (LPS) and IFN-γ at two concentrations (100 ng/ml LPS[4-6] and 10 ng/ml IFN-γ[6, 7] or 250 ng/ml LPS[2] and 20 ng/ml IFN-γ[2, 5]) (Table 1). Potential M2 macrophages were incubated in RPMI medium containing 20 ng/ml IL-4[2, 4-6]. Positive control samples (M0-like macrophages) were cultured in medium without any cytokines. After 48 hours of stimulation[2, 7], medium was collected, and cells were harvested for gene expression analysis.