Mammalian Cell culture Techniques

Preparation of Mammalian Cell Culture and Subculture


Once cell lines received from the source, it should to be checked for the contamination and confluency under the inverted microscope in sterile conditions. Ensure the required complete medium is available and cell culture room is sterile. If the cell are not confluent (30%), T flask should be kept in the Co2 incubator for 12h with 5% CO2 at 37°C with >90% humidity supplement.


  1. Cell culture lab should be sterile, wipe laminar air flow chamber with 70% isopropyl alcohol and keep UV light at least for 20 min before starting cell culture work.
  2. Prepare complete medium (10%) – 10% FBS + 1% L-Glutamine + 1% Penicillin Streptomycin + Media (Make up to required amount) under laminar air flow.
  3. Autoclave the water if media is available in the form of powder and add along with the supplements in the laminar air flow.
  4. Ensure the growth of the cell lines in T flask at least two times in day under the inverted microscope.
  5. If confluency is reached 70%, then prepare for subculture.


  1. Remove the old media from the flask and discard in beaker. Wash the T flak with PBS at least two times.
  2. Add 1 mL of Trypsin- EDTA and keep the flak at 37°C for 10 min.
  3. Gently shake the flask under the laminar air flow until the cell gets detached. Check the progress in the Inverted microscope.
  4. If cells are detached, aspirate the medium from the flask in the centrifuge tube. Add 5 mL fresh media and centrifuge at 1000 rpm for 10 min.
  5. After centrifugation clear pallet will be visible in the bottom of the tubes.
  6. Remove the supernatant carefully without disturbing the pallet.
  7. Add fresh medium up to 5 mL to remove the traces of Trypsin- EDTA.
  8. Again centrifuge it at 1000 rpm for 10 min.
  9. Discard the supernatant, and add required amount of fresh medium.
  10. Suspend the pallet.
  11. For subculture add 5mL of complete medium in the T flask (25-cm² flask or 75-cm² ) and add prepared culture of the cells from the centrifuge tube.
  12. 1 flask can be split in 3 to 5 flasks.

Remaining amount of the suspended cell culture can be preserved in Liquid Nitrogen with the help of cryo-preservant.