SUBCULTURING PROTOCOL

Prepare to subculture the cells when the culture has reached around 80% confluence.

The following protocol is for adherent cells; for suspension cells, start at step 6.

Protocol

1. Pre-warm the complete growth media given by the mobile line datasheet at a 37oC
   water bath. Pre-warm the Trypsin-EDTA (TM050) to room temperature.
2. Carefully aspirate the culture media in the culture vessel without bothering the
   cell monolayer.
3. Insert pre-warmed Trypsin-EDTA to the culture vessel. Gently rock the culture vessel
   To ensure complete coverage of this Trypsin-EDTA over the cells. (Table 1 provides
   Guidelines to the volumes of Trypsin-EDTA needed for a range of culture vessels).
4. Celebrate the cells under a microscope to confirm They’re dissociating from each
   other and are rounding up. Gently tap the culture vessel from several sides to
   promote cell detachment. Cells that are difficult to detach can be put in 37oC for
   Several moments to facilitate dispersal.
5. When majority of the cells have detached, add an equal quantity of the complete
   Media into the culture vessel to neutralize the trypsin-EDTA. Gently swirl or pipette
   The culture suspension to ensure the neutralization is complete.
6. Transfer the culture suspension into a sterile centrifuge tube.
7. Centrifuge the cell suspension at 1500 rpm for 3 minutes. The actual centrifuge
   Duration and speed may vary depending upon the cell type.
8. Aspirate the supernatant after checking all cells are pulled down into the pellet.
   Re-suspend the cell pellet in pre-warmed fresh complete media (adjust volume as
   necessary according to Table 1).
9. Pre-warm new culture vessels to 37 °C*. Seed cells in the recommended seeding density.
10. Set the newly seeded culture vessel at a 37oC*, 5% CO2 incubator. Incubate for at least
    24 – 48 hours before processing the cells for downstream experiments.
11. Renew the culture media every 2-3 days if the cells have not attained 80% confluency.
    Table 1: Volume of Trypsin-EDTA and Culture Media for Different Culture Vessels
    Culture Wares Trypsin-EDTA Volume Complete Media Volume
    12- well plate 0.5 ml/ well 2.0 ml/ well
    6- well plate 1.0 ml/ well 3.0 ml/ well
    T-25 flask 3.0 ml/ flask 7.0 ml/ flask
    T-75 flask 5.0 ml/ flask 20.0 ml/ flask
    100 mm dish 3.0 ml/ dish 10.0 ml/ dish
    150 mm dish 5.0 ml/ dish 20.0 ml/ dish