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Human ADRA1A/Galpha15 Stable Cell Line-Chem-1

Cat.No.: CSC-RG0604
Cell Line Description: This human alpha1A-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant alpha1A 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 alpha1A and its ligands.
Background: The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the alpha- and beta-adrenoceptors. The three members of the alpha1 subclass of adrenoceptors, alpha1A, alpha1B and alpha1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue; the alpha1A subtype plays a prominent role in urogenital smooth muscle contraction and renal artery contraction. Activation of alpha1 adrenoceptors also influences cell proliferation; alpha1A inhibits cell growth by arresting progression at the G1/S transition. The alpha1A subtype undergoes alternative splicing to generate four variants that differ at their C-termini, although these variants appear to be functionally identical.
Growth Properties: Adherent
Host Cell: Chem-1
Cell Line Validation: 1. Gene expression: qPCR experiments determined specific silencing of ADRA1A.2. Protein expression: ADRA1A in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by A61603: ~ 2.3 nM.
Application: Calcium flux assay, ligand binding assays
Sub-type: Adrenoceptors
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: ADRA1A adrenergic, alpha-1A-, receptor [ Homo sapiens ]
Official Symbol: ADRA1A
Synonyms: ADRA1A; adrenergic, alpha-1A-, receptor; ADRA1C; alpha-1A adrenergic receptor; ADRA1L1; alpha-1A adrenoceptor; alpha-1A adrenoreceptor; G protein coupled receptor; alpha-1C adrenergic receptor; adrenergic, alpha-1A-, receptor variant 1; adrenergic, alpha-1A-, receptor variant 3; adrenergic, alpha-1A-, receptor variant 5; adrenergic, alpha-1A-, receptor variant 8; ALPHA1AAR;
Gene ID: 148
mRNA Refseq: NM_000680
Protein Refseq: NP_000671
MIM: 104221
UniProt ID: P35348
Chromosome Location: 8p21.2
Pathway: Adrenoceptors, organism-specific biosystem; Amine ligand-binding receptors, organism-specific biosystem; Calcium Regulation in the Cardiac Cell, organism-specific biosystem; Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (12/13) signalling events, organism-specific biosystem;
Function: G-protein coupled receptor activity; adrenergic receptor activity; alpha1-adrenergic receptor activity; alpha1-adrenergic receptor activity; alpha1-adrenergic receptor activity; receptor activity; signal transducer activity;

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