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Figure 1. Calcium flux in D2-expressing Chem-7 cell line induced by Quinpirole. D2-expressing Chem-7 cells and Wild-Typ...
Figure 2. Assay for antagonist activity at D2 by calcium flux assay. D2-expressing Chem-7 cells were loaded with Fluo-4. Raclopride (D2-like...
ChemiScreen™ Calcium-Optimized Stable Cell Line Human Recombinant D2 Dopamine Receptor
Description:
ChemiScreen™ Calcium-Optimized Stable Cell Line Human Recombinant D2 Dopamine Receptor
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Product Overview:
Full-length human DRD2 cDNA encoding D2
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). Chemicon's cloned human D2L-expressing cell line is made in the Chem-7 host, which supports high levels of recombinant D2 expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between D2 and its ligands.
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:
SPECIFICATIONS:
EC50 for calcium mobilization by Dopamine: ~ 76 nM
EC50 for calcium mobilization by Quinpirole: ~ 29 nM
IC50 for Raclopride with 2x EC50 Dopamine: 0.22 nM
IC50 for SCH23390 with 2x EC50 Dopamine: 831 nM
EC50 for calcium mobilization by Dopamine: ~ 76 nM
EC50 for calcium mobilization by Quinpirole: ~ 29 nM
IC50 for Raclopride with 2x EC50 Dopamine: 0.22 nM
IC50 for SCH23390 with 2x EC50 Dopamine: 831 nM
Cell Type:
Chem-7
Cell Line Type:
GPCR Cell Lines
Host Cells:
Chem-7, an adherent cell line expressing a promiscuous G-protein.
Format:
Cell Line
Presentation:
Cells are frozen at 2 x 106 cells/mL in F-12K Nutrient Mixture, Kaighn's Modification /20% heat inactivated fetal bovine serum /10% DMSO. Cell line tests negative for mycoplasma.
Storage Conditions:
1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years.
2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C 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 37°C 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 37°C 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 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 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 Plating Media for plating for calcium assay.
2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C 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 37°C 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 37°C 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 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 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 Plating Media for plating for calcium assay.
Gene Symbol:
- DRD2
- D
- D2R
- D2DR
Protein or Enzyme Type:
GPCRs
Materials Required but Not Delivered:
Chem-7 Growth Media:
F-12K Nutrient Mixture, Kaighn's Modification containing 2 mM L-glutamine (Invitrogen 21127)
10% heat-inactivated FBS
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
G-418 (250ug/mL) (from 50 mg/mL stock, Invitrogen 10131-027)
Zeocin (200 ug/mL) (from 100 mg/mL stock, Invitrogen 45-0430)
Chem-7 Plating Media:
F-12K Nutrient Mixture, Kaighn's Modification
10% heat-inactivated FBS
1x Pen-Strep
Chem-7 Freezing Media:
F-12K Nutrient Mixture, Kaighn's Modification
20% heat-inactivated FBS
1x Pen-Strep
10% DMSO (cell culture grade)
10% heat-inactivated FBS
100 U/mL Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
G-418 (250ug/mL) (from 50 mg/mL stock, Invitrogen 10131-027)
Zeocin (200 ug/mL) (from 100 mg/mL stock, Invitrogen 45-0430)
Chem-7 Plating Media:
F-12K Nutrient Mixture, Kaighn's Modification
10% heat-inactivated FBS
1x Pen-Strep
Chem-7 Freezing Media:
F-12K Nutrient Mixture, Kaighn's Modification
20% heat-inactivated FBS
1x Pen-Strep
10% DMSO (cell culture grade)
Concentration:
2 x 106 cells/mL