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Human CASR/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0623
Cell Line Description: This 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 Galpha15 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.
Background: 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 calcium. Allosteric "calcimimetic" potentiators are being evaluated for treatment of primary hyperparathyroidism.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of CASR. 2. Protein expression: CASR in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by CaCl2: ~ 0.11mM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Calcium sensor
Propagation: Complete growth medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x Nonessential amino acids + 10mM HEPES + 1x Pen-Strep + 250μg/mL Genetecin/G-418 Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-StrepAtmosphere: air, 95%; carbon dioxide (CO2), 5%Temperature: 37 °C
Starting Cells From Frozen Cell Stock: 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.
Subculturing: 1. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca2+ and Mg2+ (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 Growth Media per 1 mL trypsin. 2. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Freezing medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 20% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep + 10% DMSO
Storage: Liquid nitrogen
Preservation: 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Subculture-Step 1). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10^6 cells/mL in Freezing Media (cell densities of 2-10 x 10^6 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. 2. 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.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: CASR calcium-sensing receptor [ Homo sapiens ]
Official Symbol: CASR
Synonyms: CASR; calcium-sensing receptor; HHC, HHC1, hypocalciuric hypercalcemia 1; extracellular calcium-sensing receptor; FHH; GPRC2A; NSHPT; severe neonatal hyperparathyroidism; parathyroid Ca(2+)-sensing receptor 1; parathyroid cell calcium-sensing receptor; CAR; FIH; HHC; EIG8; HHC1; PCAR1; MGC138441;
Gene ID: 846
mRNA Refseq: NM_000388
Protein Refseq: NP_000379
UniProt ID: P41180
Chromosome Location: 3q21.1
Pathway: Class C/3 (Metabotropic glutamate/pheromone receptors), organism-specific biosystem; E-cadherin signaling in keratinocytes, organism-specific biosystem; G alpha (i) signalling events, organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class C Metabotropic glutamate, pheromone, organism-specific biosystem;
Function: G-protein coupled receptor activity; phosphatidylinositol phospholipase C activity; protein binding; receptor activity; signal transducer activity;

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