Apr 29, 2022

Public workspaceThawing of CT-2A cell line

This protocol is a draft, published without a DOI.
  • 1MDC Berlin
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Protocol Citation: Guido Krähenbühl 2022. Thawing of CT-2A cell line. protocols.io https://protocols.io/view/thawing-of-ct-2a-cell-line-b8gurtww
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: April 29, 2022
Last Modified: April 29, 2022
Protocol Integer ID: 61684
Abstract
This protocol describes the thawing of mouse CT-2A glioma cells

Guidelines
GMO safety level 1
Materials
ReagentDMEMThermo Fisher ScientificCatalog #41966
ReagentFBSInvitrogen - Thermo Fisher

Protocol materials
ReagentDMEMThermo Fisher ScientificCatalog #41966
Materials
ReagentFalcon 15 mL Polystyrene Conical TubeFisher ScientificCatalog #352095
In 2 steps
ReagentPetri dish, 10cm, polystyreneFisher ScientificCatalog #FB0875712
Step 6
ReagentFBSInvitrogen - Thermo Fisher
Materials
Before start
Make sure to warm up media to Temperature37 °C

Remove cryovial from liquid nitrogen storage and place on dry-ice for transfer to cell culture lab.


Required
Cryovial IDs
Additional Identifiers

Remove cryovial from dry-ice and hold in a Temperature37 °C waterbath until thawed
Safety information
Avoid submerging the lid or rim of the cryovial in the waterbath as this may result in contamination

Dry the cryovial thoroughly, spray with 70% ethanol and transfer to cell culture hood
Transfer contents of cryovial to a ReagentFalcon 15 mL Polystyrene Conical TubeFisher ScientificCatalog #352095 containing Amount7 mL DMEM, 10% FBS, no antibiotics .

Rinse the cryovial with Amount1 mL of the cell suspension and add it back to the same ReagentFalcon 15 mL Polystyrene Conical TubeFisher ScientificCatalog #352095

Centrifuge at Centrifigation300 rcf, Room temperature, 00:05:00

5m
Centrifigation
Discard supernatant, resubstitute with fresh Amount7 mL DMEM, 10% FBS, no antibiotics
Transfer cell suspension to a ReagentPetri dish, 10cm, polystyreneFisher ScientificCatalog #FB0875712 and agitate gently back and forth and side to side to evenly distribute cells
Place the petri dish in a Temperature37 °C Incubator (5%CO2)

After 24 hours, inspect adherence and confluency.
Expected result



Note
If a major amount of cells did not attach, remove the media and replace with Amount7 mL DMEM, 10% FBS, no antibiotics and check for surviving cells after 24 hours