KC-4219

293T-CRE-Luc2-VPAC2-Cell-Line

42805

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Background of 293T-CRE-Luc2-VPAC2-Cell-Line

Vasoactive intestinal peptide (VIP) receptors are a group of Gs-protein-coupled receptors that are currently divided into two subtypes; VPAC1 and VPAC2. VPAC1 and VPAC2 have diverse biological actions including promotion of neuronal survival, regulation of glycogen metabolism, stimulation of prolactin release from the pituitary and catecholamine release from the adrenal medulla, and inhibition of IL-2-mediated T-cell proliferation. The human genes encoding VPAC1 and VPAC2 receptor have been located on chromosomes 3p22 and 7q36.3 respectively.

Specifications

Catalog Number	KC-4219
Cell Line Name	293T-CRE-Luc2-VPAC2-Cell-Line
Host Cell Line	293T-CRE-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of VPAC2 signaling pathway
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Cell model for monitoring VPAC2 signaling pathway
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +150μg/ml Hygromycin+1μg/ml Puromycin
Selection Marker	Hygromycin and Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

Characterization

Figure: 293t-cre-Luc2-vpac2 cell line was seeded into the 96-well plate, and treated with PACAP(1-38) at a maximum concentration of 1000ng/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/ml Hygromycin B and 1μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Jan Fahrenkrug, Edward J. Goetzl, et al. VIP and PACAP receptors in GtoPdb v.2023.1. IUPHAR/BPS Guide to Pharmacology CITE. 2023 Apr;(1).
Alexander SPH, Christopoulos A, Davenport AP, et al. The Concise Guide to PHARMACOLOGY 2023/24: G protein-coupled receptors. Br J Pharmacol. 2023 Oct;180 Suppl 2:S23-S144.