Phorbol myristate acetate (pma)

The concentrated samples were divided into two aliquots and one of the aliquots was treated with 12.5 μL of PMA (2 mM; Biotium, Inc., Hayward, CA) to a final concentration of 25 μM [80 (link)], followed by thorough mixing and incubation in the dark for 5 min at room temperature. Samples were then exposed to light with the PhAST blue-photoactivation system for tubes (GenIUL, S.L., Terrassa, Spain) for 15 min [81 (link)]. Information deduced from PMA-treated samples was documented for viable microbial population and data derived from the PMA-untreated aliquot was reported as total (dead and live) microbial population. Both, the PMA-treated and non-treated samples were further split in half, with one half subjected to bead beating with the Fastprep-24 bead-beating instrument (MP Biomedicals, Santa Ana, CA). The samples were run at 5 m/s for 60 s. After bead beating, the samples were combined with their respective analog, which were not subjected to bead beating, and the DNA from the combined sample was extracted by the Maxwell-16 MDx automated system according to the manufacturer’s instructions (Promega, Madison, WI). The purified DNA was eluted into a final volume of 50 μL.

MCs and neutrophils (1 × 10⁶ cells/well) were incubated in 24-well plates pretreated with fibronectin (1 mg/well; Life Technologies) for 3 h. Adherent cells were stimulated with 100 µg/ml LPS, 25 nM phorbol-12-myristate-13-acetate (PMA) (MP Biomedicals), or bacteria (bacterium-to-cell ratio of 25 and 50 bacteria per cell for S. aureus and C. burnetii, respectively) at 37°C. The roles of CD36 and TLR4 were studied using MCs pretreated with 5 µg/ml CD36-blocking antibodies (Abs) (mouse IgG2a; Thermo Fisher Scientific) or 10 µg/ml of the TLR4 inhibitor polymyxin B sulfate (Sigma-Aldrich) for 10 min.