Aug 22, 2024
Version 2
Thawing Frozen Adherent Cell Lines V.2
- 1Washington University
Protocol Citation: Carolina Lopez 2024. Thawing Frozen Adherent Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xz3klqe/v2Version created by Carolina Lopez
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: August 21, 2024
Last Modified: August 22, 2024
Protocol Integer ID: 106116
Keywords: thawing frozen adherent cell line, frozen adherent cell lines this protocol, adherent cell lines this protocol, adherent cell line, cell, procedure, line
Abstract
This protocol describes the procedures from thawing and expanding adherent cell lines
Materials
TISSUE CULTURE MEDIUM (TCM): Filter through 0.2 µm filter
| Component | Amount | Conc. Supp. | Product information | |
| DMEM | 500 mL | Gibco Cat. # 11965092 (#11965118-cs) | ||
| Gentamicin | 500 µL | 50µg/mL | Gibco Cat #15750060 (#15750078-pk) – 50 mg/mL | |
| Sodium Pyruvate | 5.0 mL | 1mM | Corning Cat #25-000-Cl – 100mM | |
| L-Glutamine | 5.5 mL | 2 mM | Sigma Aldrich Cat #G7513 – 200mM | |
| FBS | 50 mL | 10% |
Mycoplasma Removal Agent (MRA) - MP Biomedicals. Cat # 093050044
Protocol
Day 1
- Prepare media ahead of time and warm to room temperature
- Spray down each bottle with 70% Ethanol before placing it into the TC hood
- Pre-cool the centrifuge to 4ºC
- Prepare and label a 6-well plate, and aliquot 9 mL of TCM into a 15 mL conical for each tube of cells to be thawed
- Find the location of the cells you will be thawing in the liquid nitrogen inventory google doc
- Transfer the cells between the liquid nitrogen and the TC room on ice
- Record the information on the cryotube – date, number of cells, etc
- Begin adding media from the 15mL conical to the still frozen cells, then transfer media back and forth between the two tubes until the cells have completely thawed and everything has been transferred to the 15 mL conical
- Spin down the cells at 1200rpm for 10’ at 4ºC
- Pour off the supernatant and break up the cell pellets by tapping the tubes
- Add 1 mL of TCM to the cells, pipette up and down to break up any clumps, add 9 mL TCM, and transfer 1-2 mL of resuspended cells to a 6-well plate (cells should reach 80% confluency in two days)
- Add 10 µl of Mycoplasma removal agent per 1 mL of media
- Confirm the presence of cells in the well by microscope
- Incubate at 37ºC
Day 3 *If cells tested negative for mycoplasma before freezing, only 1 round of MRA treatment is necessary. If mycoplasma presence is questionable or unknown 2 rounds of MRA treatment should be done – see mycoplasma protocol for more information.
- Warm media to room temperature
- Remove media and wash cells once with PBS
- Add 1 mL of 0.05% Trypsin-EDTA to wells and incubate plate at 37ºC for 1-3 minutes until cells are completely released from the plate
- Add 4 mL of media to wells to halt trypsinization and transfer to a T25 flask
- Incubate at 37ºC
Day 4
- Warm media to room temperature
- Check for cell adherence
- If there are a lot of dead cells, change the media on the cells
- If cells are nearing 50% confluence, trypsinize, transfer in 10 mL to a T75 flask – Label as passage #0