Jun 05, 2019
Version 2
Thawing and Seeding Frozen Cells V.2
- 1University of Arizona
- Yoon Lab
Protocol Citation: Kenneth Schackart 2019. Thawing and Seeding Frozen Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.3rcgm2w
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: 24068
Abstract
How to thaw cells from the liquid nitrogen storage and seed into 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
(1) T-75 or T-25 flask per frozen cell vial (or more if plating at a lower density)
(1) 15 mL centrifuge tube per frozen cell vial
(1) 10 mL serological pipet tip per T-75 flask or (1) 5 mL serological pipet tip per T-25 flask
Warmed cell culture media
1000 μL filter pipette tips
Before start
- Warm cell culture media, DPBS, and Trypsin-EDTA in 37 °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 00:15:00 .
Thaw Cells
Thaw Cells
Thaw cells by suspending cryotube in 37 °C water bath until completely thawed, but no longer than necessary
Transfer cell suspension
Transfer cell suspension
Within biosafety cabinet, transfer cell suspension to 15 mL centrifuge tube using 1000 μL pipette.
Dilute freezing medium
Dilute freezing medium
Add 1 mL warmed cell culture medium to cell suspension dropwise.
Note
Adding the inital cell culture medium slowly helps prevent cell death caused by a rapid change in osmotic pressure.
Add an additional 3 mL warmed cell culture medium to cell suspension slowly.
Centrifuge cell suspension
Centrifuge cell suspension
Centrifuge the cell suspension at 1500 rpm for 00:03:00 .
Resuspend Cells
Resuspend Cells
Remove bulk of supernatant with serological pipet, then remove remainder with 1000 μL pipette.
Note
For small cell pellets, you are better off leaving a small amount of media than disturbing the cell pellet.
Add 1 mL warmed cell culture media to cell pellet.
Note
Allowing the cell pellet rest in media for about 2 minutes will help with resuspension.
Gently pipette mix the cell pellet into the solution.
Add an additional 7 mL warmed cell culture media [3 mL for T-25].
Seed Cells
Seed Cells
Using a serological pipet, transfer the cell suspension to the tissue culture flask.
Label Flask
Label Flask
Label the flask with:
- Cell line
- Passage number
- Date
- Your intials
Incubate
Incubate
Transfer flask to CO2 incubator.
Documentation
Documentation
Don't forget to remove the vial you used from the frozen storage inventory.