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Calcium flux in CaS-expressing Chem-1 cell line induced by CaCl2. CaS-expressing Chem-1 cells and Wild-Type Chem-1 cells (Chemicon catalog...
ChemiScreen™ Human Recombinant CaS Calcium Sensor Calcium-Optimized Stable Cell Line
Description:
ChemiScreen™ Human Recombinant CaS Calcium Sensor Calcium-Optimized Stable Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Full-length human CASR cDNA encoding CaS
Background Information:
Calcium homeostasis in vertebrates is regulated by hormonal communication between the parathyroid and thyroid glands, kidney, bone, and intestine. A central mediator of this regulatory system is the calcium sensor CaS (also known as CaR), a class III GPCR that is activated by high concentrations of extracellular calcium and other divalent cations. The cell types that are responsive to variations in blood calcium concentration express CaS and respond to changes in extracellular calcium in a CaS-dependent fashion. Activation of CaS in parathyroid chief cells inhibits PTH release, which results in decreased calcium mobilization from bone. In contrast, stimulation of CaS activity in the C cells of the thyroid increases secretion of calcitonin, which causes decreased bone resorption and increased urinary excretion of calciuum. Allosteric "calcimimetic" potentiators are being evaluated for treatment of primary hyperparathyroidism (Brown and MacLeod, 2001). Chemicon’s cloned human CaS-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CaS expression on the cell surface and contains high levels of the promiscuous G protein G15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CaS and its ligands.
Application Notes:
Calcium flux assay, ligand binding assays
Species Reactivity:
Human
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.
Entrez Gene Summary:
The calcium-sensing receptor (CASR) functions as a sensor for parathyroid and kidney to determine the extracellular calcium concentration and thus helps to maintain a stable calcium concentration. Mutations that inactivate CASR cause familial hypocalciuric hypercalcemia, whereas mutations that activate CASR are the cause of autosomal dominant hypocalcemia. An altenatively spliced transcript variant encoding 1088 aa has been found for this gene, but its full-length nature has not been defined.
UniProt Summary:
FUNCTION: SwissProt: P41180 # Senses changes in the extracellular concentration of calcium ions. The activity of this receptor is mediated by a G- protein that activates a phosphatidylinositol-calcium second messenger system.
SIZE: 1078 amino acids; 120674 Da
SUBUNIT: Interacts with VCP and RNF19A.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Found in kidney, but not in brain, lung, liver, heart, skeletal muscle, or placenta.
PTM: N-glycosylated. & Ubiquitinated by RNF19A; which induces proteasomal degradation.
DISEASE: SwissProt: P41180 # Defects in CASR are the cause of familial hypocalciuric hypercalcemia type 1 (FHH) [MIM:145980]; in which the receptor has reduced activity. FHH is characterized by altered calcium homeostasis. Affected individuals exhibit mild or modest hypercalcemia, relative hypocalciuria, and inappropriately normal PTH levels. & Defects in CASR are the cause of neonatal severe primary hyperparathyroidism (NSHPT) [MIM:239200]; in which the receptor has reduced activity. NSHPT is a rare autosomal recessive life- threatening disorder characterized by very high serum calcium concentrations, skeletal demineralization, and parathyroid hyperplasia. In some instances NSHPT has been demonstrated to be the homozygous form of FHH. & Defects in CASR are the cause of autosomal dominant hypoparathyroidism (FIH) [MIM:146200]. FIH is characterized by hypocalcemia and hyperphosphatemia due to inadequate secretion of parathyroid hormone. Symptoms are seizures, tetany and cramps.
SIMILARITY: SwissProt: P41180 ## Belongs to the G-protein coupled receptor 3 family.
SIZE: 1078 amino acids; 120674 Da
SUBUNIT: Interacts with VCP and RNF19A.
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Found in kidney, but not in brain, lung, liver, heart, skeletal muscle, or placenta.
PTM: N-glycosylated. & Ubiquitinated by RNF19A; which induces proteasomal degradation.
DISEASE: SwissProt: P41180 # Defects in CASR are the cause of familial hypocalciuric hypercalcemia type 1 (FHH) [MIM:145980]; in which the receptor has reduced activity. FHH is characterized by altered calcium homeostasis. Affected individuals exhibit mild or modest hypercalcemia, relative hypocalciuria, and inappropriately normal PTH levels. & Defects in CASR are the cause of neonatal severe primary hyperparathyroidism (NSHPT) [MIM:239200]; in which the receptor has reduced activity. NSHPT is a rare autosomal recessive life- threatening disorder characterized by very high serum calcium concentrations, skeletal demineralization, and parathyroid hyperplasia. In some instances NSHPT has been demonstrated to be the homozygous form of FHH. & Defects in CASR are the cause of autosomal dominant hypoparathyroidism (FIH) [MIM:146200]. FIH is characterized by hypocalcemia and hyperphosphatemia due to inadequate secretion of parathyroid hormone. Symptoms are seizures, tetany and cramps.
SIMILARITY: SwissProt: P41180 ## Belongs to the G-protein coupled receptor 3 family.
Species:
Human
Quality Assurance:
EC50 for calcium mobilization by CaCl2 ~ 0.11mM
Cell Type:
Chem-1
Cell Line Type:
GPCR Cell Lines
Brand Family:
Chemicon
Host Cells:
Chem-1
Presentation:
Cells are frozen at 2 x 106 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Protein Target:
CaS
UniProt Number:
Ligand Type:
non-peptide
Target Sub-Family:
Calcium sensor
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 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 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 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-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 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 Plating Media for plating for calcium assay.
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GPCR Class:
C
Packaging:
2 x 106 cells/vial
Gene Symbol:
- CASR
- PCAR1
- GPRC2A
- FHH
- CAR
- MGC138441
- FIH
- HHC1
- NSHPT
- HHC
- CaSR
Protein or Enzyme Type:
GPCRs
Product Name:
ChemiScreen™ Human Recombinant CaS Calcium Sensor Calcium-Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
Chem-1 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)
250μg/mL Genetecin/G-418
Chem-1 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-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
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)
250μg/mL Genetecin/G-418
Chem-1 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-1 Freezing Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)