| Field | Specification |
|---|---|
| Mfr No | |
| Species |
The Hep-70.4 hepatoma cell line is derived from a mouse liver tumor, specifically from the C57BL/6J mouse strain. This cell line is notable for its mutations in the p53 gene, which were identified at different passages during in vitro propagation. At passage number 8, a weak additional signal was detected in the single-strand conformation polymorphism (SSCP) analysis, indicating the presence of a p53 mutation. By passage number 38, two distinct p53 point mutations were identified: a G:C to C:G transversion at codon 135 and a C:G to G:C transversion at codon 138 of exon 5. These mutations led to amino acid changes from alanine to proline and cysteine to tryptophan, respectively.
The Hep-70.4 cell line displays a morphological phenotype that varies significantly during its propagation. Some sublines exhibit an epithelial morphology, while others show a fibroblast-like appearance. This heterogeneity reflects the complex nature of the cell line and its adaptability under different culture conditions. The presence of both normal and mutated p53 alleles in the early passages suggests that the mutations confer a selective growth advantage, leading to the predominance of mutated clones over time.
Intermediate filament protein analysis of the Hep-70.4 cell line revealed the expression of simple keratins K8 and K18, which are typical of normal liver cells, as well as vimentin and keratin K19 to varying degrees. These protein patterns confirm the hepatocytic origin of the cell line and its classification as a hepatoma line. The genomic stability of Hep-70.4 was further assessed through DNA fingerprint analysis, which did not reveal any major structural abnormalities, although changes in the relative intensities of certain bands were observed with increasing passage numbers.
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- Protein expression: Keratin 8, Keratin 18, Vimentin.
- Tumorigenic: Yes, in C57BL/6J mice
- Mutational profile: p53mut (codon 277 in exon 8 => Arginin -- Threonin).
- cultureMedium: DMEM, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 3.7 g/L NaHCO3, w: 1.0 mM Sodium pyruvate (Cytion article number 820300a)
- supplements: Supplement the medium with 10% FBS
- dissociationReagent: Accutase
- subculturing: Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium.
- seedingDensity: 1 x 104 cells/cm2
- fluidRenewal: Every 3 to 5 days
- postThawRecovery: After thawing, plate the cells at 5 x 104 cells/cm2 and allow the cells to recover from the freezing process and to adhere for at least 24 hours.
- freezeMedium: As a cryopreservation medium, 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.
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