Mar 27, 2024
CMT-93 Cell Culture Protocol
- 1CBM Severo Ochoa
External link: https://www.atcc.org/products/ccl-223
Protocol Citation: Laura Gómez 2024. CMT-93 Cell Culture Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62k1dgqe/v1
Manuscript citation:
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: In development
We are still developing and optimizing this protocol
Created: March 18, 2024
Last Modified: March 27, 2024
Protocol Integer ID: 96872
Keywords: CMT-93, cell line, epithelial cell line
Abstract
CMT-93 is a cell line exhibiting epithelial morphology that was isolated from the rectum of a mouse with polyploid carcinoma.
Image Attribution
Guidelines
Working with cell cultures requires a laminar flow cabinet. It has to be radiated with UV light, cleaned with any highly effective terminal disinfectant (such as Tego‱ 2000 or Suredis‱) and 70% ethanol. All material introduced into the cabinet must also be sprayed with ethanol.
Once the work is finished, we must clean the cabinet with the detergent, then with 70% etOH and turn on the UV light for 30 min.
Materials
Plasticware:
p60 cell culture plates
p100 cell culture plates
Cell culture flasks, 75 cm2, treated for cell attachment.
15 and 50 mL centrifuge tubes
Cryovials
To prepare the complete medium:
DMEM 1X
Fetal bovine serum heat inactivated (FBS)
Glutamine 200 mM
To subculture the cells:
Trypsin-EDTA
PBS 1X
Safety warnings
Every reagent must be sterile in order to avoid contaminations.
Before start
Clean and prepare the laminar flow cabinet, turn on the water bath and warm up the culture media.
Preparation of complete growth medium (DMEM+)
Preparation of complete growth medium (DMEM+)
Add 445 mL 1X DMEM, 50 mL FBS and 5 mL glutamine 200 millimolar (mM) to a sterile 500 mL bottle and homogenize
Label the bottle with name, group, phone number, date and additions.
Close with parafilm and store at 4 °C .
Cell thawing procedure
Cell thawing procedure
Remove one vial of cell stock from the liquid nitrogen tank with gloves and forceps. Transfer them to the cell culture laboratory in an appropiate container or a box with ice.
Thaw the vial by gently shaking it in a 37 °C water bath. Thawing should be rapid (approximately 2 min).
Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol
Transfer the contents of the vial to a centrifuge tube containing 9 mL of complete culture medium and1200 rpm, Room temperature, 00:05:00
5m
Resuspend with 10 mL DMEM+ and distribute on 2 P60 plates.
Incubate cultures at 37 °C , 5% CO2 Overnight
5m
Subculturing procedure
Subculturing procedure
Remove and discard culture medium.
Rinse with PBS 1X solution and discard
Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor. Discard.
Add 2 mL of Trypsin-EDTA solution to flask and incubate 00:10:00 at 37 °C to facilitate detachment from the plate.
Note
To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
10m
Observe cells under an inverted microscope until cell layer is dispersed.
Note
If the cells are not detached already, incubate 00:05:00 more at 37 °C .
Add 5 mL of DMEM+ and aspirate cells by gently pipetting. Pour the existing volume down the walls of the flask in order to drag and collect as many cells as possible.
Collect the cell suspension in a centrifugue tube and 1200 rpm, Room temperature, 00:05:00 .
5m
Discard the supernatant into a beaker with 70% EtOH or 10% bleach.
Resuspend in medium according to the dilution to be made.
Note
A subcultivation ratio of 1:4 to 1:10 is recommended
Add 1 mL of the cell supension to new culture vessels containing 14 mL DMEM+.
Incubate cultures at 37 °C , 5% CO2 Overnight
5m
Cryopreservation and storage procedure
Cryopreservation and storage procedure
1d
Repeat steps of Subculturing procedure until the "Resuspend in medium according to the dilution to be made" step.
Resuspend in medium taking into account that for every p100 we can storage up to 2 cryovials of cells, containing 1 mL .
Prepare the cryovials with 50 µL DMSO.
Add 950 µL of the cell suspension to every cryovial.
Label the cryovials with cell line, passage, date and lab number or phone number.
Store the cryovials in a slow freezing container at -80 °C for 24:00:00 .
1d
Transfer the cryovials to the liquid nitrogen tank.
Protocol references