2.9. DAPI Test and Mycoplasma Culture on Agar

Human osteosarcoma cells (MG63) and mouse fibroblasts cells (L929) were detached using a 0.5 g/L trypsin 1:250 and 0.2 g/L Versen (EDTA) solution (Lonza, Walkersville, MD, USA) and immediately separated into single cells by pipetting. Next, the growth medium with FBS added (1:1 ratio) to stop trypsin activity. The received cellular suspension was centrifuged for 3 min at 500 RPM. The cell sediment rinsed with Dulbecco’s Phosphate-Buffered Saline (DPBS) with calcium and magnesium, and after centrifugation, the pellet was suspended in a working solution of 1 μg/mL DAPI (AppliChem Gmbh, Darmstadt, Germany Germany) in methanol and incubated at 37 °C for 10 min. Cells were rinsed with DPBS and examined under an inverted Nikon microscope (Nikon Instruments Europe BV, Amstelveen, The Netherlands) using a DAPI filter (emission at: 435–485 nm and excitation: 340–380 nm wavelength) at 400× magnification.

We have mentioned above, MG63 and L929 72 h old cells with the same cell number concentration and cell dilution ranges, staring from 1 × 10⁴ number of cells/mL (1×) to inoculate Mycoplasma Agar Base supplemented with mycoplasma selective supplement-G (MABG) (Oxoid Ltd., Hampshire, England) to test mycoplasma growth. Then, 1 mL of undiluted (1×), 10⁻¹ (10×) and 10⁻² (100×) diluted cell suspension were added into MABG plates in two independent repeats. Agar plates incubated at 37 °C in 5% CO₂ and evaluated after 1 and 2 weeks. MABG was prepared according to the manufacturer’s instructions. Evaluation of bacterial growth was performed after one and two weeks periods under direct detection using manual magnifier (~20× magnification) and inverted microscope using 400× microscope (Olympus, Hamburg, Germany) magnification.