KC-4138

293T-CRE-Luc2-RAMP1-CALCR Cell Line

59418

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Background of 293T-CRE-Luc2-RAMP1-CALCR Cell Line

Cyclic adenosine monophosphate (cAMP) is a second messenger involved in cell signaling that regulates variousl physiological and pathological processes. cAMP regulates the transcription of target genes by activating proteinl kinase A (PKA) and the transcription factor cAMP response element-binding protein (CREB). CRE is the target of many extracellular and intracellular signaling pathways, including cAMP, calcium,l GPCR (G-protein coupled receptors), and neurotrophins. The CAMP/PKA/CREB signaling pathway has both tumor-suppressive and tumor-promoting effects in cancer cells and can be useful in studying cancer signaling pathways.CALCR encodes a high affinity receptor for the peptide hormone calcitonin and belongs to a subfamily of seven transmembrane-spanning G protein-coupled receptors. The encoded protein is involved in maintaining calcium homeostasis and in regulating osteoclast-mediated bone resorption. Polymorphisms in this gene have been associated with variations in bone mineral density and onset of osteoporosis.RAMP1 is a member of the RAMP family of single-transmembrane-domain proteins, called receptor (calcitonin) activity modifying proteins (RAMPs).The RAMP1 protein is involved in the terminal glycosylation, maturation, and presentation of the CGRP receptor to the cell surface.

Specifications

Catalog Number	KC-4138
Cell Line Name	293T-CRE-Luc2-RAMP1-CALCR Cell Line
Clone Number	1#-1
Host Cell Line	293T-CRE-Luc2 Cell Line
Description	Stable 293T cell line expressing exogenous luciferase under the control of CRE responsive element and RAMP1-CALCR fusion sequence.
Quantity	One vial of frozen cells (5×10⁶ per vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 150μg/mL Hygromycin B + 1μg/mL puromycin
Selection Marker	Hygromycin B and Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-CRE-Luc2-RAMP1-CALCR Cell Line was generated using a lentiviral vector expressing the RAMP1-CALCR sequence.

Characterization

Figure 1: 293T-CRE-Luc2-RAMP1-CALCR cell were seeded into 96-well plates, treated with Calcitonin in different concentrations for 16 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin B and 1μg/mL Puromycin) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70%DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature. 1998 May 28;393(6683):333-9. doi: 10.1038/30666. PMID: 9620797.
Gorn AH, Lin HY, Yamin M, Auron PE, Flannery MR, Tapp DR, Manning CA, Lodish HF, Krane SM, Goldring SR. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. J Clin Invest. 1992 Nov;90(5):1726-35. doi: 10.1172/JCI116046. PMID: 1331173; PMCID: PMC443230.