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Calcium flux in D4-expressing Chem-5 cell line. D4-expressing Chem-5 cells and Wild-Type Chem-5 cells (Millipore catalog # HTSCHEM-5) were loaded wit...
ChemiSCREEN™ Human Recombinant D4 Dopamine Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant D4 Dopamine Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Proprietary plasmid pHS containing DRD4.4 cDNA. The stable clonal cell line was selected by resistance to geneticin and hygromycin, 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.
Background Information:
The neurotransmitter dopamine is involved in a wide variety of neuroendocrine, emotional, cognitive and locomotor functions. These activities of dopamine are mediated by a group of 5 G protein coupled receptors, 2 of which (D1 and D5) couple to Gs and 3 of which (D2, D3, D4) couple to Gi (Missale et al., 1998). The D4 receptor is highly polymorphic, particularly in the third cytoplasmic loop, which contains from 2 to 7 repeats of 16 amino acids. Individuals with 7 repeats have increased risk of developing ADHD (Thapar et al., 2005). Mice engineered to lack D4 have reduced locomotor activity, increased sensitivity to drugs of abuse that elevate dopamine (cocaine, methamphetamine and ethanol), and reduced response to novelty (Rubinstein et al., 1997; Dulawa et al., 1999). Millipore’s cloned human D4-expressing cell line is made in the Chem-5 host, which supports high levels of recombinant D4 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 D4.
Application Notes:
Calcium flux assay, ligand binding assays
Usage Statement:
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.
Species:
Human
Quality Assurance:
Table I. Comparison of EC50 values of D4-expressing Chem-5 cells with values described in the literature.
| ligand | assay | potency (nM) | Reference |
|---|---|---|---|
| Dopamine | calcium | EC50 = 1.7 | Figure 1 |
| Quinpirole | calcium | EC50 = 27.6 | Figure 1 |
| Dopamine | Inhibition of cAMP | EC50 = 3.0 | Chemal et al., 2006 |
| Quinpirole | Inhibition of cAMP | EC50 = 9.7 | Chemal et al., 2006 |
| L-745,870 | calcium | IC50 = 2.2 | Figure 2 |
| L-745,870 | GTPyS binding | IC50 = 1 | Millan et al., 1998 |
Brand Family:
Chemicon
Host Cells:
Chem-5
Presentation:
Cells are frozen at 2 x 106 cells/mL in 90% fetal bovine serum/10% DMSO. Cell line tests negative for mycoplasma.
Protein Target:
D4
Ligand Type:
non-peptide
Storage Conditions:
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-5 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-5 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-5 Plating Media for plating for calcium assay.
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-5 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-5 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-5 Plating Media for plating for calcium assay.
Target Sub-Family:
Dopamine
Promotional Text:
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GPCR Class:
A
Packaging:
2 x 106 cells/vial
Product Name:
ChemiSCREEN™ Human Recombinant D4 Dopamine Receptor Calcium-Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
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
250 μg/mL Hygromycin B
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)
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
250 μg/mL Hygromycin B
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)