Mts colorimetric cell viability kit

To confirm that the previous observations were a consequence of interference of the compounds with the differentiation process rather than a reduction in monocyte viability, THP-1 monocytes were incubated with different concentrations of the compound for 30 h. No difference in cell viability was detected. Moreover, the cytotoxic effects of the drugs on differentiated macrophages were evaluated. Following the incubation of THP-1 cells with 25 nM PMA for 24 h and 100 ng/ml LPS for 24 h, the differentiated macrophages were treated with different concentrations of the compounds for 24 h. Cell viability was assessed using MTS colorimetric cell viability kit (Abcam, Cambridge, UK).

Monocytes were cultured and differentiated as described previously³⁹ (link). THP-1 cells (human acute monocytic leukaemia lineage, American Type Culture Collection, Manassas, VA) were seeded at a density of 20 × 10⁵ cells/ml. A set of cells were exposed to 25 nM of phorbol myristate-acetate (PMA, Calbiochem, Darmstadt, Germany) for 24 h to drive differentiation. Another set was similarly treated with PMA followed by 100 ng/ml of lipopolysaccharide (LPS, invivogen, San Diego, CA, USA) for 72 h. After incubation, the supernatant was aspirated and the density of the adherent cells was estimated using MTS colorimetric cell viability kit (Abcam, Cambridge, UK). The inhibitory actions of different compounds on the differentiation process were evaluated by pre-incubation with different concentrations of each compound for 6 h. Cell viability after treatment was normalised to the reading after PMA exposure following a 6-h incubation with DMSO. All experiments were conducted in triplicates. A positive control for the effect of COX1/COX2 inhibition on monocyte-to-macrophage differentiation was obtained by treatment with diclofenac. IC₅₀ values for each compound were determined by non-linear regression as the best fit values of the log [inhibitor] vs. response curve using GraphPad Prism software.