Description

Organism – Mouse , tissue – Embryo , disease – Teratocarcinoma , , , synonyms – ATDC-5 , 129 – age – Embryo , Male – , , – Polygonal , – , , Adherent , 1 , – DMEM:Ham’s F12, w: 3.1 g/L Glucose, w: 1.6 mM L-Glutamine, w: 15 mM HEPES, w: 1.0 mM Sodium pyruvate, w: 1.2 g/L NaHCO3 (Cytion article number 820400a), – Supplement the medium with 5% FBS , , For routine adherent cell culture: Aspirate the old culture medium from the adherent cells, and wash them with PBS to remove any remaining medium. After aspirating the PBS, add the appropriate volume of Accutase solution based on the culture vessel size (e.g., 1 ml for a T25 flask, 3 ml for a T75 flask) and incubate at room temperature or 37°C for 5-10 minutes, or until the cells detach. Monitor detachment under a microscope, and gently tap the vessel if necessary to release the cells. Once detached, add complete medium to inactivate the Accutase, gently resuspend the cells, and transfer an aliquot of the cell suspension into a new culture vessel containing fresh medium. Place the vessel in an incubator set to 37°C with 5% CO2, and change the medium every 2-3 days. – , – , CM-1 (Cytion catalog number 800100) – , Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit. 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. 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. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening. 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. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium. 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. 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.