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The HK EGFP-LaminA/H2B-mCherry cell line is a genetically engineered Hela Kyoto derived cell model developed to facilitate advanced studies in nuclear dynamics and chromatin organization within living cells. This cell line expresses two fusion proteins: EGFP (enhanced green fluorescent protein) fused with Lamin A, and mCherry (a red fluorescent protein) fused with Histone H2B. The EGFP-Lamin A fusion highlights the nuclear envelope and allows for the visualization of nuclear architecture changes during cell cycle progression or under various experimental conditions. Meanwhile, the H2B-mCherry fusion protein binds to DNA and provides a vivid red fluorescence that marks chromatin, enabling real-time observation of chromosomal processes during mitosis and interphase.
These cells are invaluable for real-time imaging applications, including studies on nuclear integrity, DNA replication, and cellular aging, as well as research into diseases where nuclear architecture is disrupted, such as cancer and laminopathies. The dual-color fluorescence feature of this cell line allows for simultaneous visualization of both the nuclear envelope and chromatin, facilitating a comprehensive understanding of nuclear-cytoplasmic interactions and the spatiotemporal organization of chromatin. Such capabilities make it a critical tool for molecular biology research and cellular biophysics, providing insights into the mechanics of gene expression regulation, nuclear organization, and the cell cycle.
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- Protein expression: EGFP-LaminA/H2B-mCherry
- Products: Histone H2B
- 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: 2 to 3 times per week
- 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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