KC-3728

NCI-H69-SLC6A2-Flag-Cell-Line

34672

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Background of NCI-H69-SLC6A2-Flag-Cell-Line

SLC6A2, also called NET, is a monoamine transporter and is responsible for the sodium-chloride (Na + /Cl − )- dependent reuptake of extracellular norepinephrine, which is also known as noradrenaline. NETs, along with the other monoamine transporters, are the targets of many antidepressants and recreational drugs. In addition, an overabundance of NET is associated with ADHD. There is evidence that single-nucleotide polymorphisms in the NET gene may be an underlying factor in some of these disorders

Specifications

Catalog Number	KC-3728
Cell Line Name	NCI-H69-SLC6A2-Flag-Cell-Line
Host Cell Line	NCI-H69
Description	Stable NCI-H69 cell line expressing exogenous C-terminal Flag-tagged human SLC6A2 gene
Quantity	One vial 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 + 1μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Floating aggregates
Subculture	Split saturated culture 1:2-1:3 every 2-3 days; seed out at about 3-5 × 10⁵ cells/ml
Incubation	37 °C with 5% CO₂
Storage	37 °C with 5% CO₂
Doubling Time	Approximately 56 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

NCI-H69 SLC6A2 Flag Cell Line was generated using a lentiviral vector expressing the human SLC6A2 sequence.

Characterization

Figure: Characterization of Flag tag overexpression in NCI-H69 SLC6A2 Flag stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 1μg/mL Puromycin)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 1:2-1:3 every 2-3 days; seed out at about 3-5 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Schroeter S, Apparsundaram S, Wiley RG, Miner LH, Sesack SR, Blakely RD (May 2000). "Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter". The Journal of Comparative Neurology. 420 (2): 211–32. doi:10.1002/(SICI)1096-9861(20000501)420:23.0.CO;2-3. PMID 10753308. S2CID 24643588.
2. Tellioglu T, Robertson D (November 2001). "Genetic or acquired deficits in the norepinephrine transporter: current understanding of clinical implications". Expert Reviews in Molecular Medicine. 2001 (29): 1–10. doi:10.1017/S1462399401003878. PMID 14987367. S2CID 30965333.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.