Culture and Passage Cells

Culture and passage cells

• ready to passage when cell confluence to 80% or 90% or when the cell density reaches an average of 2 X 105 cells/cm2

• remove medium by aspiration

• wash cells with PBS to remove the residual medium

• add Trypsin-EDTA (enough to cover the flask surface)

• incubate at 37oC for 5 minutes

• observe by microscope to see cell death

• add 2ml of medium trypsinization

• pipet to detach all cells

• transfer cell suspension to 15 ml tube

• add cell culture medium to new flask

• dilute as appropriate into the flask and mix well

• a seeding density of 4.0 X 104 to 5.0 X 104 viable cells/cm2 is used when subculturing capan-1 cells

• for T-75 use 3 x 106 cells, 100 mm Petri plate (57 cm2 surface area) use 2.3 X106 cells , 60 mm Petri plate (22.1 cm 2 surface area) use 8.8 X 10 5 cells.

• incubate at 37oC

• replace media 2-3 days