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.