Jun 05, 2019

Public workspaceMammalian Cell Culture: Subculturing V.2

Version 1 is forked from Mammalian Cell Culture: Subculturing
DOI
dx.doi.org/10.17504/protocols.io.3q4gmyw
  • 1University of Arizona
  • Yoon Lab
Kenneth Schackart
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DOI: dx.doi.org/10.17504/protocols.io.3q4gmyw
Protocol Citation: Kenneth Schackart 2019. Mammalian Cell Culture: Subculturing. protocols.io https://dx.doi.org/10.17504/protocols.io.3q4gmyw
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: June 04, 2019
Last Modified: June 05, 2019
Protocol Integer ID: 24060
Abstract
This protocol details how to subculture/passage nearly confluent mammalian cells grown in a tissue culture flask.
Guidelines
  • Gloves must be worn at all times.
  • Perform all tasks within biosafety cabinet.
  • Anything entering biosafety cabinet must be generously sprayed with 70% ethanol (even you).
  • When finished, wipe biosafety cabinet with 70% ethanol, and UV for at least 15 minutes.
Materials
  • Cultured T-75 [or T-25] flask
  • Gloves
  • 0.05% or 0.25% Trypsin-EDTA
  • Cell culture Media (e.g. DMEM:F12, EMEM)
  • DPBS
  • 15 mL centrifuge tube
  • Serological pipet and tips
  • 1000 μL pipette and tips
  • Waste beaker
Safety warnings
Gloves must be worn at all times. Perform all work within biosafety cabinet.
Before start
  • Warm cell culture media, DPBS, and Trypsin-EDTA in Temperature37 °C water bath.
  • Wash waste beaker with soap and warm water, then dry with paper towel.
  • Expose serological pipet tips, centrifuge tube, and waste beaker to UV for at least Duration00:15:00 .
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
Remove media from flask.
Using serological pipette, add Amount4 mL DPBS to flask. [Amount1 mL for T-25]



Using serological pipette, remove DPBS and dispose into waste 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 Amount4 mL warmed trypsin-EDTA to T-25 flask. [Amount1 mL for T-25]


Wait approximately Duration00:05:00 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.

Note
For cell types that take longer to detach, place flask in incubator to keep the temperature high enough for trypsin to remain active.

Add Amount4 mL cell culture media. [Amount1 mL for T-25]
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 Amount9.5 mL fresh cell culture media to T-25 flask, this will preserve any remaining cells.

Centrifuge the cell suspension on Centrifigation1500 rpm for Duration00:03:00 .


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 Amount1000 µL cell culture media to the cell pellet, and allow to sit for Duration00:01:00 .
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.

Seed 2 flasks each with Amount500 µL cell suspension.
Note
You may seed more than 2 flasks, just use smaller volumes in each.



Label flask with updated passage number along with the date.
Incubate
Incubate
Incubate at Temperature37 °C in CO2 incubator.