1. Discard Old Medium: Remove the old medium from the flasks.
2. Wash Cells: Add 3-5 ml of PBS (without calcium and magnesium) to T25 flasks, or 5-10 ml to T75 flasks, to wash the adherent cells.
3. Add EDTA Solution: Cover the cell layer completely with a freshly prepared 0.05% EDTA solution?use 1-2 ml for T25 flasks and 2.5 ml for T75 flasks.
4. Incubation: Incubate the flasks at 37 degrees Celsius for 10 minutes.
5. Add Trypsin/EDTA Solution: Following the incubation, add a freshly prepared trypsin/EDTA solution (0.05% trypsin, 0.025% EDTA) to the flasks, ensuring the cells are fully covered?use 1 ml for T25 flasks and 2.5 ml for T75 flasks.
6. Monitor Detachment: Observe the cells, which should detach within 1-2 minutes.
7. Neutralize Trypsin: Add FBS-containing cell culture medium to stop the trypsin activity.
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. Optionally, skip centrifugation but remove any remaining freezing medium after 24 hours.

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.

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.