ChemiSCREEN™ Human Recombinant APJ Apelin Receptor Calcium-Optimized Stable Cell Line

ChemiSCREEN™ Human Recombinant APJ Apelin Receptor Calcium-Optimized Stable Cell Line

Chemicon (Millipore)

2 vials

Proprietary plasmids containing truncated human AGTRL1 cDNA encoding APJ and a promiscuous G protein. The stable clonal cell line was selected by resistance to geneticin and hygromyocin, followed by limited dilution cloning. The cell line was tested and found to have equivalent EC50 and signal at 1, 3 and 6 weeks of continuous culture.

Apelin peptides have been discovered to be a family of peptides of different sizes that is derived from the N-terminus of a 77 amino acid precursor peptide (preproapelin) (Hosoya et al., 2000). Apelin receptor (APJ) is a G protein-coupled receptor that is activated by several apelin fragments, which results in inhibition of cAMP production (Habata et al., 1999). APJ and apelin peptides have been found to be involved in the regulation of cardiovascular function (Katugampola et al., 2001) and fluid homeostasis (Reaux et al., 2001). Broad roles of apelin system has been established in lowering blood pressure, as a potent cardiac inotrope, in modulating pituitary hormone release and food and water intake, in stress activation, and as a novel adipokine that is excreted from fat cells and regulates insulin (Lee et al., 2006). Millipore’s cloned human APJ-expressing cell line is made in the Chem-5 host, which supports high levels of recombinant APJ expression on the cell surface and contains optimal levels of promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at APJ.

Calcium flux assay, ligand binding assays

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Human

Table I. Comparison of EC50 values of APJ-expressing Chem-5 cells with values described in the literature.

ligand	assay	potency (nM)	Reference
Apelin-13	calcium	EC50 = 5.7 - 9.3	Figure 1
Apelin-13	calcium	EC50 = 22.4	Medhurstet al., 2003
Apelin-13	Inhibition of forskolin-induced cAMP	EC50 = 0.047	Medhurst et al., 2003
Apelin-13	Inhibition of forskolin-induced cAMP	EC50 = 0.17	Habata et al., 1999
[³H]-[Radioligand binding	Kd = 2.7	Medhurst et al., 2003
[¹²⁵I]-(Pyr¹)-Apelin-13	Radioligand binding	Kd = 0.33 - 0.35	Katugampola et al., 2001
[¹²⁵I]-[Glp⁶⁵,Nle⁷⁵, Tyr⁷⁷]-Apelin-13	Radioligand binding	Kd = 0.4	Millipore HTS068M, APJ Membrane Preparation

Chemicon

1. Cells propagated for screening should be maintained and seeded at less than 90% confluency. Trypsinize cells as above and seed cells in 96-well black-walled, clear bottom plate at 50,000 cells/well in Chem-1 Plating Media. Keep the plate at room temperature for 1 h to allow even cell distribution in the plate, then transfer plate to a humidified incubator at 37ºC with 5% CO₂.

2. Chem-1 derived cell lines have been successfully assayed using multiple commercially-available calcium dye kits following the manufacture’s protocols. The protocol described below is a suggested protocol that can be generally applied to most calcium dyes kits.

3. Remove media

4. Wash cells with buffered salt solution

5. Add 100 μL/well calcium dye-loading solution.

6. Incubate the plate for 30 minutes in a humidified incubator at 37ºC with 5% CO₂.

7. Incubate the plate for an additional 60 min at 25ºC with 5% CO₂.

8. Set-up FLIPR to dispense 50uL/well 3X ligand to appropriate wells in the assay plate. Set excitation wavelength at 470-495 nm (FLIPR TETRA) or 485 nm (FLIPR1, FLIPR2, FLIPR3) and emission wavelength at 515-565 nm (FLIPR TETRA) or emission filter for Ca2+ dyes (FLIPR1, FLIPR2, FLIPR3). Set pipet tip height at 95 uL and dispense rate to 25 μL/sec. Set up plate layout and tip layout for each individual experiment. Set time course for 180 seconds, with ligand addition at 10 seconds.

9. Ligands are prepared in a white nonbinding surface 96-well plate (Corning 3605).

10. After the run is complete, negative control correction is applied and data analyzed utilizing the maximum statistic.

Chem-5

Cells are frozen at 2 x 10⁶ cells/mL in 90% fetal bovine serum/10% DMSO. Cell line tests negative for mycoplasma.

APJ

P35414

peptide

1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen.

2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37ºC with 5% CO₂.

3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.

4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca⁺⁺ and Mg⁺⁺ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC with 5% CO₂ until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Glutamate Receptor Growth Media per 1 mL trypsin.

5. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.

6. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10⁶ cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 10⁶ are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.

7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Glutamate Receptor Plating Media for plating for calcium assay.

Apelin

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A

2 x 10⁶ cells/vial

ChemiSCREEN™ Human Recombinant APJ Apelin Receptor Calcium-Optimized Stable Cell Line

NP_005152

Chem-5 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020-A)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250 μg/mL Genetecin/G-418
500 μg/ml Hygromycin


Chem-5 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep

Chem-5 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)