Elisas

The possibility of direct collagen production by macrophages was tested in primary human macrophages cultures. The cells were treated with ATP vesicles (1 and 10 mM), Mg-ATP (1 mM), lipid vesicles (1 mM) or cream 24 h post plating and for another 24/48 h. The cell culture supernatant was collected and stored at -80°C until further analysis. Collagen type 1-α-1 levels were determined from these culture supernatants through enzyme-linked immunosorbent assays (ELISAs) (My Biosource LLC, San Diego, CA, United States) in 96-well plates according to manufacturer’s protocol. All samples were evaluated in triplicate. Calibration was made using the standard curve generated by the collagen standard provided in the kit.

To assess mRNA expression by quantitative RT-PCR, cells were seeded 5 × 10⁶ cells per flasks. Next day cells were treated with control or 10 mM acetate for 24 h. Cells were harvested, and total mRNA was extracted using RNeasy kit (Qiagen, USA) according to manufacturer's instructions. RT reactions were performed using SuperScript™ IV First-Strand Synthesis System (ThermoFisher Scientific, USA) using 1 μg/mL RNA concentration and used in RT-PCR. Quantitative PCR reactions were performed using TaqMan™ Universal Master Mix II, no UNG (ThermoFisher Scientific, USA) (n = 5) using Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, USA). Taqman gene expression assays (ThermoFisher Scientific, USA) were used for β-actin (Hs01060665_g1), ACSS1 (Hs00287264_m1), and ACSS2 (Hs00218766_m1).
To assess protein levels of ACSS1 and ACSS2, cells were seeded 6 × 10⁶ cells per flask. The following day cells were treated with control or 10 mM acetate for 24 h (n = 3). Cells were collected and total protein was extracted after three freeze thaw cycles. ELISAs (MyBioSource, USA) were performed according to manufacturer's instructions and read at 450 nm using SPECTROStar Nano.

A previously characterized P. aeruginosa pneumonia model (47 (link)) was used as follows. Anesthetized mice (thiopental at 5% [wt/vol], i.p.) were infected by intratracheal instillation, using 50 μl of the MLD₁₀₀ calculated earlier. Mice remained in a vertical position for 3 min and then were left resting in 30° positions until they awakened. After 4 h of infection, 36 mice (18 under normoxia and 18 under hypoxia) were sacrificed (with sodium thiopental; Braun Medical, USA) for analysis, and 46 mice (20 under normoxia, 18 under hypoxia, and 8 under 6 h of hypoxia followed by normoxia) were analyzed at the time of death. Survival rates were analyzed for the different conditions. Bacteremia levels and bacterial loads in blood and tissues (spleen and lungs) were determined as described above. HIF-1α levels in mouse serum were measured by ELISAs (MyBioSource, USA).