Karyotype of bone marrow-derived mesenchymal stem cells
When the cell density reached 80%, colcemid was used to stop the cell cycle. Changes in cell morphology were monitored by inverted microscopy. After cell separation with trypsin, centrifugation was performed (400 g for 10 min). 5 ml of 0.075 M KCL solution with hepes was carefully and slowly added and then incubated in C37 º for 30 min. After centrifugation (300 g × 5 min), 5 ml of fixative solution containing acetic acid and methanol mixture (methanol: acetic acid ) was added to the cell pellet. The centrifugation was performed again with the previous speed and time. The fixative solution was discarded and the samples were washed twice in cold (5 ° C) fresh methanol: acetic acid solution (1: 2) to improve metaphase quality. The slides were incubated at 60 ° C for at least 16 hours to obtain G-bands. They were then immersed in trypsin solution (0.002 g /ml) for 5 seconds and washed in saline solution and finally washed rapidly with distilled water. Staining was performed using Giemsa (6: 1) dye (Trypsin and Giemsa) and the bands quality was evaluated.