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Cyclic AMP assay with D2L-expressing CHO cell line. D2L-expressing CHO cells and wild-type CHO (WT) were preincubated in 1 mM IBMX for 5 min, then ex...
ChemiSCREEN™ Human Recombinant D2L Dopamine Receptor cAMP -Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant D2L Dopamine Receptor cAMP -Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Plasmid pcDNA3 containing DRD2 long isoform cDNA encoding D2L. The stable clonal cell line was selected by resistance to geneticin, 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:
Dopamine is a catecholamine neurotransmitter that functions in the CNS to control locomotor, cognitive, emotional and neurendocrine processes, and in the periphery to modulate cardiovascular, renal and gastrointestinal processes. The biological activities of dopamine are mediated by a family of five GPCRs. The D1 and D5 subtypes couple to Gs to increase intracellular cAMP, whereas the D2, D3 and D4 subtypes couple to Gi to reduce cAMP (Missale et al., 1998). The D2 dopamine receptors have been of particular clinical interest due to their regulation of prolactin secretion and their affinity for antipsychotic drugs. The D2 receptor exists as two alternatively spliced isoforms differing in the insertion of a stretch of 29 amino acids in the third intracellular loop (D2S and D2L) (Giros et al., 1989; Grandy et al., 1989). Millipore’s cloned human D2L-expressing cell line is made in the CHO host, which supports optimal levels of recombinant D2L expression for robust agonist-induced cAMP signal. Thus, the cell line is an ideal tool for screening for agonists and antagonists at the D2L Receptor.
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 D2L-expressing CHO cells with values described in the literature.
| ligand | assay | potency (nM) | Reference |
|---|---|---|---|
| Quinpirole | cAMP | EC50 =38 | Figure 1 |
| Quinpirole | cAMP | EC50 = 14 | Moreland et al., (2004) |
Brand Family:
Chemicon
Host Cells:
CHO (Chinese hamster ovary cells)
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:
D2L
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 CHO 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 CHO 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 CHO 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 CHO 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 CHO 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 CHO 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 D2L Dopamine Receptor cAMP -Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
CHO Growth Media:
F12-K containing 2 mM L-glutamine (Invitrogen 21127)
10% heat-inactivated FBS
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250μg/mL Genetecin/G-418
CHO Plating Media:
F12-K containing 2 mM L-glutamine (Invitrogen 21127)
10% heat-inactivated FBS
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
CHO Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)
F12-K containing 2 mM L-glutamine (Invitrogen 21127)
10% heat-inactivated FBS
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250μg/mL Genetecin/G-418
CHO Plating Media:
F12-K containing 2 mM L-glutamine (Invitrogen 21127)
10% heat-inactivated FBS
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
CHO Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)