| Medio de cultivo |
RPMI 1640, w: 2.0 mM stable Glutamine, w: 2.0 g/L NaHCO3 (Cytion article number 820700a) |
| Suplementos |
Supplement the medium with 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 1% NEAA. Add Geneticin (G418-Sulfat) to achieve a final concentration of 1 mg/mL. |
| Reactivo de disociación |
Trypsin-EDTA |
| Tiempo de duplicación |
approx. 24-36 hours |
| Subcultivo |
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 Trypsin/EDTA 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 until the cells detach (5-10 minutes). Monitor detachment under a microscope, and gently tap the vessel if necessary to release the cells. Once detached, add complete medium to inactivate the Trypsin/EDTA, 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. |
| Relación de división |
1 to 5 |
| Densidad de siembra |
2 to 4 x 104 cells/cm2 |
| Renovación de fluidos |
2 to 3 times per week |
| Recuperación tras el deshielo |
After thawing, split the cells at a ratio of 1:2 to 1:3 in T25 flasks and allow the cells to recover from the freezing process and to adhere for at least 24 hours. For best attachment and viability after thawing the cells, we recommend using Collagen-coated flasks or plates for the initial seeding after cryo-recovery. Collagen coating is not required for subsequent routine culture of the cells. |
| Medio de congelación |
As a cryopreservation medium, we use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress. |
| Descongelación y cultivo de células |
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.
|
| Atmósfera de incubación |
37°C, 5% CO2, humidified atmosphere. |
| Condiciones de envío |
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. |
| Condiciones de almacenamiento |
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. |
