CMT-93 Cell Culture Protocol

Add 445mL 1X DMEM, 50mL FBS and 5mLglutamine 200millimolar (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.

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 9mL of complete culture medium and1200rpm

Resuspend with 10mL DMEM+ and distribute on 2 P60 plates.

Incubate cultures at 37°C, 5% CO₂0h 5m 0s

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 2mL of Trypsin-EDTA solution to flask and incubate 0h 10m 0s at 37°C to facilitate detachment from the plate.

Observe cells under an inverted microscope until cell layer is dispersed.

Add 5mL 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 1200rpm.

Discard the supernatant into a beaker with 70% EtOH or 10% bleach.

Resuspend in medium according to the dilution to be made.

Add 1mL of the cell supension to new culture vessels containing 14mL DMEM+.

Incubate cultures at 37°C, 5% CO₂0h 5m 0s

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 1mL.

Prepare the cryovials with 50µL DMSO.

Add 950µL 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 24h 0m 0s.

Transfer the cryovials to the liquid nitrogen tank.