Evaluating Bone Powder and Oxytetracycline Effects on K562 Cells

The human lymphoblast derived K562 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured at 37 °C in a humidified atmosphere (5% CO₂) in RPMI (Sigma–Aldrich, St. Louis, Missouri, USA) medium for 48 h. Oxytetracycline (OTC) from Sigma–Aldrich (St. Louis, Missouri, USA) was stocked at 100 mM in DMSO (Sigma–Aldrich, St. Louis, Missouri, USA). Bone powder, achieved from poultry raised with or without the administration of oxytetracycline, was extracted in cell culture medium as previously described [19 (link),20 (link)]. Briefly, the bone powder was dissolved in RPMI at the concentration of 124 mg/mL and kept under continuous stirring at 37 °C for 48 h. After that, the suspension was filtered, and the filtrate neutralized with KOH (Sigma–Aldrich, St. Louis, Missouri, USA) at pH 7.2–7.4. For compound treatment, 0.5–2.0 × 10⁶ cells/mL were supplemented with graded concentration of OTC or graded dilution of bone powder extracts as indicated. The viability of K562 cells was assessed before and after treatments by direct counting Trypan Blue-negative cells with a hemocytometer.