Jul 06, 2020

Public workspaceThawing adherent cancer cell lines V.2

This protocol is a draft, published without a DOI.
  • 1Wellcome Sanger Institute
Emily Souster
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Protocol Citation: Emily Souster, Verity Goodwin, Charlotte Beaver, Adam Jackson, Fiona Behan, Rizwan Ansari, Mathew Garnett 2020. Thawing adherent cancer cell lines. protocols.io https://protocols.io/view/thawing-adherent-cancer-cell-lines-bh9tj96nVersion created by Emily Souster
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: July 06, 2020
Last Modified: July 06, 2020
Protocol Integer ID: 38931
Abstract
This SOP is for thawing an adherent cancer cell line from a frozen cryovial.

Process diagram:


Materials
MATERIALS
ReagentCell culture treated T75 flasksCorningCatalog #430641
Select an appropriate culture media for your cell line. Common culture medias used for cancer cell lines are serum supplemented Advanced DMEM F-12 or RPMI, in the presence of pen-strep.

Equipment

Pipette boy
25ml stripette
P1000 pipette and tips
Microbiology Safety Cabinet (MSC)
Light Microscope
Temperature37 °C waterbath
Temperature37 °C , 5% CO2 incubator


Safety warnings
Retrieving vials from liquid nitrogen tanks is a significant ergonomic risk due to potential weight of filled racks. To reduce the risk of manual handling injury, lifting racks out of dewers should be kept to a minimum.
Before start
  • Pre-warm complete culture media to room-temperature.

  • Add 18ml complete culture media to a T75 flask.
Remove the cryovial from liquid nitrogen storage and place on dry-ice for transfer to cell culture lab.
Take the cryovial from the dry-ice and hold in a Temperature37 °C waterbath until thawed.
Note
Be careful here to avoid submerging the lid or rim of the cryovial in the waterbath.
Dry the cryovial thoroughly, spray with 70% ethanol and transfer to cell culture hood.
Use a P1000 pipette to transfer the contents of the cryovial to the T75 flask containing Amount18 mL media.

Use Amount1 mL complete culture media to wash the cryovial and add this to the flask.

Place the flask in a Temperature37 °C , 5% CO2 incubator. Agitate the flask gently back and forth and side to side to ensure even distribution of cells across the flask.

After 24 hours, inspect adherence and confluency. Remove the media using a sterile aspirating pipette and replace with Amount12 mL complete culture media.