HeLa Kyoto EGFP-alpha-tubulin/H2B-mCherry
Marca : CLS Cell Lines Service
HK EGFP-alpha-tubulin/H2B-mCherry Cells
General information
Description | The HK EGFP-alpha-tubulin/H2B-mCherry HeLa cell line is a meticulously engineered model designed for detailed visualization of cellular processes. This clonal line has been stably transfected to express two fluorescent protein fusions that enable real-time imaging of both chromatin and the microtubular network. The red fluorescent protein mCherry is fused to the core histone protein H2B, creating H2B-mCherry. This fusion protein is expressed from the pH2B-mCherry-IRES-neo3 plasmid and serves as a chromatin marker, highlighting the nuclear DNA in live-cell imaging and facilitating studies on chromatin dynamics and nuclear architecture. Additionally, this cell line expresses monomeric enhanced GFP (Green Fluorescent Protein) fused to ?-tubulin, introduced via the pmEGFP-?-tubulin-IRES-puro2b plasmid. The GFP-?-tubulin fusion provides a vivid green fluorescence that outlines the microtubule structures within the cell. This feature is crucial for studying microtubule organization, dynamics, and their role in cell division and intracellular transport. The stable integration of these constructs allows for continuous, long-term observation of these cellular components without the need for repeated transfection, thus reducing variability and enhancing the reliability of experimental results. Drug resistance selection following transfection ensures the stability and uniformity of expression among the cells in this line. |
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Organism | Human |
Tissue | Cervix |
Disease | Carcinoma |
Synonyms | HeLa Kyoto EGFP-a-tubulin/H2B-mCherry, HeLa H2B-mRFP and mEGFP-alpha-tubulin |
Characteristics
Age | 30 years |
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Gender | Female |
Ethnicity | African American |
Morphology | Epithelial-like cells with mosaic stone shape |
Growth properties | Monolayer, adherent |
Identifiers / Biosafety / Citation
Citation | HK EGFP-alpha-tubulin/H2B-mCherry (Cytion catalog number 300670) |
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Biosafety level | 1 |
Depositor | Dr. J. Ellenberg, EMBL Heidelberg |
Expression / Mutation
Protein expression | EGFP-alpha-tubulin, H2B-mCherry: Location/Gene: 1..589 / Pcmv, 652..1029 H2B, 1042..1752 / mCherry, 2983..3777 / KanR/NeoR |
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Viruses | Negative for HIV, HBV and HCV. |
Products | CMV Promotor, Histone H2B, Neomycin, Phosphotransferase |
Handling
Culture Medium | DMEM, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 1.5 g/L NaHCO3, w: 1.0 mM Sodium pyruvate (Cytion article number 820300a) |
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Medium supplements | Supplement the medium with 10% FBS |
Passaging solution | Accutase |
Doubling time | 24 hours |
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. |
Split ratio | A ratio of 1:3 is recommended |
Seeding density | 1 x 10^4 cells/cm^2 |
Fluid renewal | 2 to 3 times per week |
Freezing recovery | After thawing, plate the cells at 5 x 10^4 cells/cm^2 and allow the cells to recover from the freezing process and to adhere for at least 24 hours. |
Freeze medium | CM-1 (Cytion catalog number 800100) or CM-ACF (Cytion catalog number 806100) |
Handling of cryopreserved cultures |
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Quality control / Genetic profile / HLA
Sterility | 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. |
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