KC-3797

293T-CRE-Luc2-RAMP2-CALCRL Cell Line

34796

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Background of 293T-CRE-Luc2-RAMP2-CALCRL Cell Line

Receptor activity-modifying proteins (RAMPs) are mammalian accessory proteins that interact allosterically with the vast majority of GPCRs. Their actions are wide-reaching and highly variable, but include modulation of receptor trafficking, changes to ligand specificity and alteration of intracellular responses to receptor activation. As RAMPs interact with their cognate GPCRs in a complex lipid membrane environment, their effects on receptor function vary depending on the cell type in which they are studied.G-protein-coupled receptors (GPCRs) have been reported to participate in the occurrence and development of avariety of human cancers. CALCR is one of the hundreds of GPCRs. Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide, which is primarily localized to C and A sensory fibers. These fibers display a wide innervation throughout the body, with extensive perivascular localization, and have a dual role in sensory (nociceptive) and efferent (effector) function.

Specifications

Catalog Number	KC-3797
Cell Line Name	293T-CRE-Luc2-RAMP2-CALCRL Cell Line
Clone Number	3#
Host Cell Line	293T-CRE-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of RAMP2 signaling pathway
Quantity	One vial of frozen cells (≥2-10⁶/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+1µg/mL Puromycin+150µg/mL Hygromycin B
Selection Marker	Puromycin \| Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-CRE-Luc2-RAMP2-CALCRL cell line was generated using lentivirus expressing RAMP2&CALCRL sequence.

Characterization

Figure 1. 293T-CRE-Luc2-RAMP2-CALCRL cell line was seed into the 96-well plate, and treated with Adrenomedullin at a maximum concentration 10μg/mL diluted 10-fold for 6 hours, then readout with Bright-Glo system.

Cell Resuscitation

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

Cell Freezing

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

References

1.McGlone ER, Manchanda Y, Jones B, Pickford P, Inoue A, Carling D, Bloom SR, Tan T, Tomas A. Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling. Mol Metab. 2021 Nov;53:101296. doi: 10.1016/j.molmet.2021.101296. Epub 2021 Jul 13. PMID: 34271220; PMCID: PMC8363841.
2.Russell FA, King R, Smillie SJ, Kodji X, Brain SD. Calcitonin gene-related peptide: physiology and pathophysiology. Physiol Rev. 2014 Oct;94(4):1099-142. doi: 10.1152/physrev.00034.2013. PMID: 25287861; PMCID: PMC4187032.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.