Feb 06, 2019
Mammalian Cell Culture: Subculturing
- 1University of Arizona
- 481b Laboratory
Protocol Citation: Kenneth Schackart, Kattika Kaarj 2019. Mammalian Cell Culture: Subculturing. protocols.io https://dx.doi.org/10.17504/protocols.io.xukfnuw
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: February 06, 2019
Last Modified: February 06, 2019
Protocol Integer ID: 20076
Abstract
This protocol details how to subculture nearly confluent mammalian cells grown in a T-25 flask.
Materials
- Gloves
- 0.05% or 0.25% warmed Trypsin-EDTA
- Warmed cell culture Media (e.g. DMEM:F12, EMEM)
- DPBS
- 15 mL centrifuge tube
- Serological pipette and tips
- 1000 μL pipette and tips
Safety warnings
Lab coat and gloves must be worn at all times.
Assess Cell Confluency
Assess Cell Confluency
Under light microscope, look at the cells and assess level of confluency. This is how you will determine the need to subculture.
Note
Confluency can be estimated by evaluating the percentage of surface covered by cells.
Low confluency SH-SY5Y
High confluency SH-SY5Y
Wash Cells
Wash Cells
Using serological pipette, add 1 mL DPBS to T-25 flask.
Using serological pipette, remove DPBS and dispose into wase beaker.
Repeat the above 2 steps, so that you will wash the cells twice.
Note
Always use a fresh pipette tip when drawing liquid from a stock solution.
Trypsinize
Trypsinize
Add 1 mL warmed trypsin-EDTA to T-25 flask.
Wait 00:05:00 for trypsin-EDTA to detach the cells.
Note
This time will vary in practice, and depends on cell type and trypsin concentration (i.e. 0.05% vs 0.25%). Some cell types may take up to 15 minutes. In those cases, assess detachment progress using a light microscope.
Add 1 mL cell culture media.
Note
Trypsin-EDTA is neutralized by adding a volume of cell culture media equal to that of trypsin-EDTA.
Spin Down
Spin Down
Using a serological pipette, transfer the cell suspension (cells, trypsin-EDTA, and cell culture media) into a 15 mL centrifuge tube.
Add 3.5 mL fresh cell culture media to T-25 flask, this will preserve any remaining cells.
Centrifuge the cell suspension on 1.5 kRPM for 00:03:00 .
Note
This has been perfomed by T.A. ahead of time. Bring your cell suspension to trade for a spun down cell pellet.
Centrifuge tubes with large cell pellets at the bottom and 8 mL of supernatant.
Resuspend and Reseed
Resuspend and Reseed
Remove supernatant, dispose into waste beaker.
Note
You can leave a small amount with the serological pipette, the rest will be taken off in the next step.
Using a 1000 μL pipette, carefully remove the remaining supernatant, being cautious not to disturb the cell pellet.
Safety information
Always dispose of pipette tips in sharps container. Do not use the same tip twice.
Add 1000 µL cell culture media to the cell pellet, and allow to sit for 00:01:00 .
Gently pipette mix the cell pellet until the pellet is resuspended.
Note
Pipette mixing is done by slowly drawing in solution and pushing it out several times, all without removing the pipette tip from the solution. Ask T.A. for help on this if you need some pointers.
Reseed your T-25 flask by transferring 500 µL cell suspension to the flask.
Note
In practice you would reseed the other 500 µL into another flask, or even split the cell suspension into 3 or 4 flasks. Alternatively, you make take the remaining cell suspension for an experiment.
Incubate
Incubate
Incubate at 37 °C in CO2 incubator.