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Human TACR3-tGFP Stable Cell Line-SHSY5Y

Cat.No.: CSC-RG0030
Cell Line Description: SHSY5Y-HuTACR3-tGFP cell line is a bone marrow neuroblastoma cell line of the human brain, which has been transfected with a Human Tachykinin receptor 3 (TACR3) tagged in the C-terminus with tGFP to allow stably express of the human TACR3 tagged in the C-terminus with tGFP protein. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: Neurokinin 3 receptor is the gene that encodes a protein that is one of the three Neurokinin receptors (NKRs), also termed as TACRs. The Neurokinin receptor family is a group of Gcoupled receptors whose principal ligands are the Neurokinins. This protein family encompasses a wide range of functions including various parachine, autocrine and endocrine processes. NK3 receptors are distributed widely throughout the CNS, and are found in high levels in the cerebral cortex, basal ganglia and dorsal horn of the spinal chord. They have a limited distribution in peripheral tissues, and are found in ganglia (e.g., myenteric plexus), kidney, and in a limited number of smooth muscles (e.g., rat portal vein). NK3 antagonist has been proposed as antipsychotics.
Growth Properties: Mixed, adherent and suspension
Morphology: Epithelial
Host Cell: SHSY5Y
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human TACR3.2. Functional validation: Activation and Internalization assay for NK3R-tGFP
Sub-type: Tachykinin
Propagation: Complete growth medium: Eagle"s Minimum Essential Medium and F12 Medium. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: 1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.
Subculturing: 1. Remove and discard culture medium.2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.3. Add 1.0 to 2.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.5. Add appropriate aliquots of the cell suspension to new culture vessels.6. Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:20 to 1:50 is recommended Medium Renewal: every 4 to 7 days
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 95%; DMSO, 5%
Storage: Liquid nitrogen
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
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: TACR3 tachykinin receptor 3 [ Homo sapiens ]
Official Symbol: TACR3
Synonyms: TACR3; tachykinin receptor 3; neuromedin-K receptor; neurokinin beta receptor; NK3R; NK-3 receptor; neurokinin B receptor; NKR; NK-3R; TAC3RL; MGC148060; MGC148061;
Gene ID: 6870
mRNA Refseq: NM_001059
Protein Refseq: NP_001050
MIM: 162332
UniProt ID: P29371
Chromosome Location: 4q25
Pathway: Calcium signaling pathway, organism-specific biosystem; Calcium signaling pathway, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (q) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem;
Function: G-protein coupled receptor activity; receptor activity; signal transducer activity; tachykinin receptor activity;

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