KC-2453

NCI-H1975-AKR1B1-Luc2 Cell Line

24092

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Background of NCI-H1975-AKR1B1-Luc2 Cell Line

AKR1B1, also known as AR, is an enzyme that is encoded by the AKR1B1 gene in humans, which belongs to the aldehyde-keto reductase superfamily, with a widely expression in human organs including the kidney, lens, retina, nerve, heart, placenta, brain, skeletal muscle, testis, blood vessels, lung, and liver. Adapting AKR1B1 inhibitors could as well prevent sepsis complications, prevent angiogenesis, ameliorate mild or asymptomatic diabetic cardiovascular autonomic neuropathy and may be a promising strategy for the treatment of endotoxemia and other ROS-induced inflammatory diseases.

Specifications

Catalog Number	KC-2453
Cell Line Name	NCI-H1975-AKR1B1-Luc2 Cell Line
Host Cell Line	NCI-H1975
Description	Stable NCI-H1975 cell line expressing exogenous luciferase gene under the control of AKR1B1 signaling pathway.
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+1µg/mL Puromycin
Selection Marker	Puromycin
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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

NCI-H1975 cell line was generated using lentivirus expressing AKR1B1 sequence.

Characterization

Figure 1. Characterization of AKR1B1 overexpression in NCI-H1975 stable clones using Western Blot and Bright-Glo readout.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 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.Graham A, Heath P, Morten JE, Markham AF (March 1991). The human aldose reductase gene maps to chromosome region 7q35. Human Genetics. 86 (5): 509–14. doi:10.1007/BF00194644. PMID 1901827. S2CID 34446965. 2.Grewal AS, Bhardwaj S, Pandita D, Lather V, Sekhon BS (2016-01-01). Updates on Aldose Reductase Inhibitors for Management of Diabetic Complications and Non-diabetic Diseases. Mini Reviews in Medicinal Chemistry. 16 (2): 120–62. doi:10.2174/1389557515666150909143737. PMID 26349493. 3.O'connor T, Ireland LS, Harrison DJ, Hayes JD (October 1999). Major differences exist in the function and tissue- specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members. The Biochemical Journal. 343 Pt 2 (2): 487 – 504. doi:10.1042/bj3430487. PMC 1220579. PMID 10510318. 4.Lefrançois-Martinez AM, Bertherat J, Val P, Tournaire C, Gallo-Payet N, Hyndman D, Veyssière G, Bertagna X, Jean C, Martinez A (June 2004). Decreased expression of cyclic adenosine monophosphate-regulated aldose reductase (AKR1B1) is associated with malignancy in human sporadic adrenocortical tumors. The Journal of Clinical Endocrinology and Metabolism. 89 (6): 3010–9. doi:10.1210/jc.2003-031830. PMID 15181092.