Method for passaging the cells
Before using, all the reagents were brought to room temperature. A 10 ml
serological pipette was used to remove media from the 80-90 percent
confluent flasks. 10 mL PBS was used to wash the cells in the T-75
flask. The flask was filled with two millilitres of 0.1 percent trypsin
EDTA. The flask was kept at 37°C in the CO2 incubator
for 2-3 min and was observed under microscope for detachment. For
trypsin inhibition, six millilitres of growth media were added to the
flask and pipetted back into place. The cell suspension was collected in
15 ml falcon tube, and then centrifuged at 1200 rpm for 3 min.
The pellet was resuspended in 3 ml of complete media after the supernatant was discarded.
Cells were counted, and then 0.2-0.4 million cells were kept in T-25
flask for growing. The flasks were incubated in CO2incubator at 37 °C and the cells were periodically monitored for any
morphological changes and contamination. After the formation of 80- 90%
confluent monolayer, the cells were further utilized.