HaCaT Cells

1. Discard Old Medium: Carefully remove the old culture medium from the flasks.
2. Wash Cells: Add 3-5 ml of phosphate-buffered saline (PBS) without calcium and magnesium to T25 flasks, or 5-10 ml to T75 flasks, to rinse the adherent cells.
3. Add EDTA Solution: Cover the cell layer entirely with a freshly prepared 0.05% EDTA solution. Use 1-2 ml for T25 flasks and 2.5 ml for T75 flasks.
4. Incubate: Incubate the flasks at 37°C for 10 minutes.
5. Add Trypsin/EDTA or TrypLE Express Solution: After incubation, add a freshly prepared trypsin/EDTA solution (0.05% trypsin, 0.025% EDTA) or TrypLE Express to the flasks, ensuring the cell layer is fully covered. Use 1 ml for T25 flasks and 2.5 ml for T75 flasks. (Note: Steps 3 and 4 can be omitted if using TrypLE Express.)
6. Monitor Detachment: Observe the cells under a microscope. The cells should detach within 1-5 minutes.
7. Neutralize Trypsin: Add cell culture medium containing fetal bovine serum (FBS) to neutralize the trypsin activity as soon as the cells have detached.
8. Transfer Cells: Dispense the cell suspension into new flasks pre-filled with fresh culture medium.

1. Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit.

2. Upon receipt, either store the cryovial immediately at temperatures below -150°C to ensure the preservation of cellular integrity, or proceed to step 3 if immediate culturing is required.

3. For immediate culturing, swiftly thaw the vial by immersing it in a 37°C water bath with clean water and an antimicrobial agent, agitating gently for 40-60 seconds until a small ice clump remains.

4. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening.

5. Carefully open the disinfected vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of room-temperature culture medium, mixing gently.

6. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium.

7. Gently resuspend the cell pellet in 10 ml of fresh culture medium. For adherent cells, divide the suspension between two T25 culture flasks; for suspension cultures, transfer all the medium into one T25 flask to promote effective cell interaction and growth.

8. Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.

37°C, 5% CO₂, humidified atmosphere.

Cryopreserved cell lines are shipped on dry ice in validated, insulated packaging with sufficient refrigerant to maintain approximately −78 °C throughout transit. On receipt, inspect the container immediately and transfer vials without delay to appropriate storage.

For long-term preservation, place vials in vapor-phase liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable only as a short interim step before transfer to liquid nitrogen.

Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods.

To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections.