The THP-1 monocyte cell line can be differentiated into macrophage-like cells upon stimulation with phorbol 12-myristate 13-acetate (PMA) and therefore widely used in inflammatory research. However, there is a lack of evidence-based protocols for cell culture and differentiation. In this study, we highlight how culture conditions influence THP-1 cells and their response to PMA stimulation. After thawing, THP-1 cells were cultured for up to 4 weeks and monitored by phase-contrast microscopy. Stressed cells (>1 × 106 cells/mL for 72 h without medium replenishment) were assessed for CD80 and MHC-II expression and the subset distribution by flow cytometry. For PMA stimulation (10 μg/mL), standard cells under normal nutrient conditions (5 × 105 cells/mL), low (2 × 105 cells/mL), and high (1 × 106 cells/mL) cell densities, along with stressed cells at 1 × 106 cells/mL density, were seeded and observed at 24 and 48 h. Cells formed aggregates containing more than 10 cells during the first week in suspension but gradually transitioned to single cells with continuous culture. Metabolically stressed cells developed protrusions, showed increased adherence, and expressed higher levels of MHC-II compared to standard cells (p = 0.02). These cells also displayed signs of partial differentiation, with fewer MHC-II−CD80− cells (p < 0.001) and more MHC-II+CD80− cells (p < 0.001) than non-stressed cells. Optimal and uniform differentiation was observed after 3–4 weeks of recovery, at seeding densities below 1 × 106 cells/mL, and under non-stressed conditions. These findings show that THP-1 culture conditions, including recovery time post-thawing, stress status and seeding density, have a major impact on their responsiveness to PMA.
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