KC-3472

NIH/3T3-Luc2 Cell Line

27105

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Background of NIH/3T3-Luc2 Cell Line

Luciferase is an oxidative enzyme that can produce bioluminescence with the adddition of luciferin, but don’t need an external light source, which is different from fluorescent proteins. The bioluminescence can be detected directly by light sensitive device, such as luminometer or modified microscope. Luciferase is widely used in many fields ofbiological research, such as transcriptional activity, kinase or other enzyme activity, cellular ATP level, whole animal imaging.

Specifications

Catalog Number	KC-3472
Cell Line Name	NIH/3T3-Luc2 Cell Line
Clone Number	3#
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 clone with Luciferase gene overexpression
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%DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:4 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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

NIH/3T3-Luc2 Cell Line was generated using a lentiviral vector expressing luciferase sequence.

Characterization

Figure 1: Characterization of luciferase expression in NIH/3T3 using the Bright-Lite Luciferase Assay System.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS )in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

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

References

1. Greer LF 3rd, Szalay AA. Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence. 2002 Jan-Feb;17(1):43-74. doi: 10.1002/bio.676. PMID: 11816060.