Doxycycline doxycycline hyclate

For the drug treatment experiments, 12Z or primary stromal endometriotic cells were seeded in 6-well plates at a density of 3 × 10⁵ cells per well for 12Z and 6 × 10⁵ cells per well for primary stromal cells. The following day, the medium was replaced by serum-free medium after washing the cells once with phosphate-buffered saline. After 24 h the medium was replaced with 2 ml of serum-free medium containing drugs in the indicated doses. Serum-free medium was used to avoid foreign protease activity in the serum as has been done by others [19 (link)] and no signs of reduced cell viability were found as described below. Doxycycline (doxycycline hyclate, Sigma-Aldrich) was purchased and stock solutions were diluted in sterile water to a concentration of 1 mg/ml and kept in the dark at − 20 °C. Treatment doses were chosen according to cell culture doses for human cell lines reported in the literature [20 (link)]. Progesterone (Sigma-Aldrich) was stored at − 20 °C as a stock solution in DMSO at a concentration of 100 mM. Drugs were freshly diluted from the stock for every treatment. Controls were treated with the vehicle substance in the same concentration.

Toxicity of doxycycline for cultured cells was established using cell proliferation reagent WST-1 (Roche, Rotkreuz, Switzerland, according to the manufacturer’s instructions. doxycycline (doxycycline hyclate, Sigma-Aldrich, St. Louis, MO, USA) was diluted to a concentration of 50 mg/mL in water and this preparation was added to the growth media (GM) to the final desired concentration (5 to 50 µg/mL). This range of doxycycline concentrations was chosen as it encompasses concentrations that are achievable in sera following administration of the recommended dose (2.5 to 10 mg/kg) to cats [43 (link)].