Basic cell culture (HEK293T)#

Cell lines#

Tatsuya’s cell line list

Reagent#

  • Cell

  • DMEM (high glucose without L-glutamine)

  • Fetal bovine serum

  • TrypLE Express

  • Penicillin/Streptomycin (P/S)

  • Cell cutlure medium

  • DMEM +10% FBS + 1%P/S

Reagent

Volume

DMEM

450 ml

FBS

50 ml

P/S

5 ml

Total

505 ml

Protocols#

Thawing cells from freezing cells

  1. (Optional) Coat new flask(s) with gelatin, and let sit for one hour in hood

    • T75: 5mL

    • 6 well plate: 1mL per well

  2. Warm DMEM (+FBS) media.

  3. Thawing freezing vial of cells (from LN2 Tank) at 37C water bath

  4. Add 8 ml of medium into new 15 ml falcon tube.

  5. Add 1 ml of cell suspention from freezing vial into 15 ml of falcon tube.

  6. Centrifuge 15ml of falcon tube at 1000 rpm for 3 min at RT.

  7. Aspirate supernatant

  8. Add 10ml of fresh clulture medium into 15 ml of falcon tube containing cell pelet. Pipette and mix for several times.

  9. Transfer cell suspensin to T75 flask (10 cm culture dish).

  10. Put T75 flask into incubator.

Medium change

  1. Aspirate culture medium from T75 flask

  2. Add 10 ml of culture medium into T75 flask

Subculture

  1. Aspirate culture medium from T75 flask

  2. Add 4 ml of DPBS into T75 flask

  3. Aspirate all DPBS from T75 flask

  4. After aspirating PBS, add 3mL of trypsin

  5. Add 3ml of TrypLE express into T75 flask

  6. Incubate and monitor for 3-5 min

  7. Add flesh 6 ml of culture medium, and then collect all soulstion into a 15mL falcon tube.

  8. Centrifuge 15 ml of falcon tube at 1000 rpm for 3 min at RT.

  9. Aspirate supernatant

  10. Add 10ml of fresh clulture medium into 15 ml of falcon tube containing cell pelet. Pipette and mix for several times.

  11. Add 2ml of cell suspension into new T75 flask (* 5, spilit ratio 1:5)

  12. Add fresh 8ml of culture medium into T75 flask.

  13. Put T75 flask into incubator.