KC-0259

HCC827-nuclear-RFP-Cell-Line

20117

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Background of HCC827-nuclear-RFP-Cell-Line

Red fluorescent protein (RFP) is a fluorescent protein separated from Discosoma that emits red fluorescent under ultraviolet and has the longest excitation wavelength and emission wavelength up to now. RFP is composed of 225 amino acids, and is widely used in animals, plants and yeast gene expression system.

Specifications

Catalog Number	KC-0259
Cell Line Name	HCC827-nuclear-RFP-Cell-Line
Host Cell Line	Human HCC827 cell line
Description	Stable HCC-827 cell line expressing exogenous RFP with nuclear localization
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+2μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

HCC-827 nuclear-RFP cell line was generated using a lentiviral vector expressing RFP with a nuclear localization sequence.

Characterization

Figure: Characterization of the expression of nuclear-localized RFP in HCC-827 stable clone using fluorescent microscope.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 2μg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Atsushi Miyawaki, Daria M Shcherbakova and Vladislav V Verkhusha. Red fluorescent proteins: chromophore formation and cellular applications. Current Opinion in Structural Biology, 22(5), 10, 2012: 679-688