| Product Description |
Recombinant CHO-K1 cells stably overexpress human vasoactive intestinal peptide receptor 2 (VPAC2) on the surface and contain high levels of G protein Gαs to couple with the receptor in downstream signaling pathways. |
| Applications |
Calcium flux assay, and cAMP accumulation assay. |
| Expressed Gene |
NCBI reference sequence NM_003382; no expressed tags |
| Target Protein |
NP_003373 |
| Host Cell |
CHO-K1 |
| Size |
Two vials of frozen cells (>1×106 per vial in 1 mL) |
| Storage |
Store cells in liquid nitrogen immediately upon receipt. Thaw and recover cells within one year from the date received. |
| Mycoplasma Status |
Negative. The mycoplasma test was performed with MycoAlert™ PLUS Mycoplasma Detection Kit (Cat. No. LT07-318, Lonza). |
| Stability |
Stable through more than 16 passages with no significant changes in assay performance or expression profile. |
| Culture Properties |
Adherent |
| Freeze Medium |
45% Ham’s F-12K (Kaighn’s) (Cat. No. 21127, Life Technologies), 45% FBS (Cat. No. 10099-141, Life Technologies), 10% DMSO (Cat. No. D2650, Sigma) |
| Complete Growth Medium |
Ham’s F-12K (Kaighn’s), 10% FBS |
| Culture Medium |
Ham’s F-12K (Kaighn’s), 10% FBS, 200 μg/ml Zeocin (Cat. No. R250-01, Life Technologies), 100 μg/ml Hygromycin B (Cat. No. 10687010, Life Technologies) |
| Gene Synonyms |
VIPR2, C16DUPq36.3, DUP7q36.3, PACAP-R-3, PACAP-R3, VIP-R-2, VPAC2R, VPCAP2R |
| Background |
The Vasoactive intestinal polypeptide receptor 2 (VPAC2) is a Gs-coupled receptor expressed in the stroma of uterus and prostate; smooth muscles in gastrointestinal tract, seminal vesicles and skin; blood vessels; thymus. VPAC2 is an essential regulator of the circadian pacemaker of the hypothalamic suprachiasmatic nuclei. |
Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with VIP in CHO-K1/VPAC2/Gα15 cells. d2 acceptor fluorophore -labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in CHO-K1/VPAC2/Gα15 cells competitively bind with Europium Cryptate-labeled anti-cAMP monoclonal antibody. The FRET signal decreases as the intracellular cAMP concentration rises and was measured by plate reader (Pherastar, BMG). The EC50 of VIP on CHO-K1/VPAC2/Gα15 cells was 6.17 nM.
Figure 1. VIP-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/VPAC2/Gα15 cells. The cells were loaded with Calcium-4 (Cat. No. R8142; Molecular Devices) prior to stimulation with VAPC2 agonist, VIP. The intracellular calcium change was measured by FLIPRTETRA. The relative fluorescent units (RFU) were recorded and normalized to plot against the log of the cumulative doses of VIP (mean ± SEM, n = 3). The EC50 of VIP on CHO-K1/VPAC2/Gα15 cells was 0.25 μM.
Notes:
EC50 value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom) / (1+10^ ((LogEC50-X)*Hill Slope))
X is the logarithm of concentration. Y is the response.
Y is % of stimulation of RFU and starts at Bottom and goes to Top along a sigmoid curve.
For research use only. Not intended for human or animal clinical trials, therapeutic or diagnostic use.
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