Protocol Citation: PMAT0001 2020. Cell subculture. protocols.io https://protocols.io/view/cell-subculture-bnk9mcz6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 20, 2020
Last Modified: October 20, 2020
Protocol Integer ID: 43393
Guidelines
- For every 25cm2 of surface area, fill with 5 mL media
- Use about 10 mL to 15 mL of media for 75cm2
- PBS used is 1X
- 10% FBS in DMEM (50 mL )
- 1% PenStrep in DMEM (5 mL )
Materials
MATERIALS
TrypsinBio Basic Inc.Catalog #TB0626.SIZE.1g
Fetal bovine serum
Dulbecco’s Phosphate Buffered SalineSigma AldrichCatalog #D5652
Penicillin StreptomycinInvitrogen - Thermo FisherCatalog #15140 122
DMEMInvitrogen - Thermo FisherCatalog #11885
Before start
- Thoroughly wipe down hood and any item introduced into the hood with 70% ethanol
- Carry out asceptic techniques while working
Pre-warm reagents to 37 °C in water bath for about 00:15:00 .
15m
Aspirate spent culture media from cell culture vessel.
Wash cells once with PBS (2 mL is enough to wash T25 flasks and maybe 5 mL for T75 flasks).
Aspirate PBS (from the side of the plate that does not have any cells, so as to avoid disturbing the cells).
Add 2 mL of Trypsin-EDTA (Add this volume for T25 flasks and accordingly about 5mL for T75 flasks) in the cell culture flasks; Incubate flasks for about 00:01:00 .
1m
After incubation, examine cells under a microscope. Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask. A few taps can be applied onto the flask to make cells detach faster.
Once the cells have detached, add FBS/serum-containing medium to the flask in an equal ratio to the added trypsin. Tyrypsin will start acting on excess serum proteins instead of harming cells.
Collect harvested cells and pipette into an appropriate-sized centrifuge tube.
Centrifuge cells for 00:05:00 at 1500rpm.
5m
After centrifuging, aspirate media and trypsin.
Add 2 mL of fresh media into the tubes.
In each of the new flask (T25), add 5 mL of fresh media.
Add about 1 mL of cells from step 11 into each of the new flasks.
Rule of Thumb for ratio of splitting
Rule of Thumb for ratio of splitting
- After the cells are 80% confluent, we should split them as they are in the log phase of growth
- Refer to attached images for the rule of splitting
Screenshot 2020-10-20 at 13.34.28.png 
Screenshot 2020-10-20 at 13.35.08.png