As a model for monocytes, the THP-1 cell line (number TIB-202l; American Type Culture Collection, Manassas, VA) was used, realizing the cell culture within RPMI 1640 culture media, which was supplemented with 10% inactivated FBS and 1% antibiotic-antimycotic (Life Technologies, Grand Island, NY).
In order to obtain activated monocytes, THP-1 cells were exposed to 1 µg/ml LPS from Escherichia coli serotype 0111:B4 (Sigma–Aldrich) for 48 h. The THP-1 cells (resting monocytes) and the activated monocytes that were obtained were seeded at a density of 7 × 105 cells/ml on a six-well plate, either without hormonal stimulus (W/S) or with hormonal stimulus (i.e. LLH (low) and HLH (high) hormone concentrations in the first trimester or third trimester). To the obtain macrophages, 3 × 106 THP-1 cells were differentiated with 200 nM phorbol myristate acetate for 3 d, followed by resting culture with fresh RPMI for 5 d.17 (link) Thereafter, the macrophages that were obtained were exposed to the same treatments as previously described for monocytes. All cell cultures were carried out in a humidified atmosphere at 37°C and CO2 5%, in triplicate for each condition.
Next, we evaluated the effect of the pregnancy hormone mixture on cytokine production and surface marker expression in resting and activated monocytes and macrophages derived from THP-1 cells with measurements detailed below.
In order to obtain activated monocytes, THP-1 cells were exposed to 1 µg/ml LPS from Escherichia coli serotype 0111:B4 (Sigma–Aldrich) for 48 h. The THP-1 cells (resting monocytes) and the activated monocytes that were obtained were seeded at a density of 7 × 105 cells/ml on a six-well plate, either without hormonal stimulus (W/S) or with hormonal stimulus (i.e. LLH (low) and HLH (high) hormone concentrations in the first trimester or third trimester). To the obtain macrophages, 3 × 106 THP-1 cells were differentiated with 200 nM phorbol myristate acetate for 3 d, followed by resting culture with fresh RPMI for 5 d.17 (link) Thereafter, the macrophages that were obtained were exposed to the same treatments as previously described for monocytes. All cell cultures were carried out in a humidified atmosphere at 37°C and CO2 5%, in triplicate for each condition.
Next, we evaluated the effect of the pregnancy hormone mixture on cytokine production and surface marker expression in resting and activated monocytes and macrophages derived from THP-1 cells with measurements detailed below.
