Tango-H_MRGPRX2 CHO-K1 Cell Line

Cat. No: GM-C37884
Product: Tango-H_MRGPRX2 CHO-K1 Cell Line

MRGPRX2 (Mas-related G protein-coupled receptor member X2) is a G protein-coupled receptor (GPCR) primarily expressed in mast cells and certain neural tissues. The MRGPRX2 receptor is involved in various physiological and pathological processes, including pain transmission, immune responses, and inflammatory reactions. It can recognize and bind to a variety of ligands, including cyclic peptides and pro-inflammatory molecules. MRGPRX2 is associated with certain allergic diseases, such as chronic urticaria and anaphylactic shock, as its expression in mast cells can trigger the release of histamine and other inflammatory mediators. Additionally, this receptor is involved in some drug-induced allergic reactions, making it a potential target of medical interest in drug development and allergy research.

Tango-H_MRGPRX2 CHO-K1 Cell Line is a clonal stable CHO-K1 cell line constructed using lentiviral technology, constitutive expression of the MRGPRX2 gene constructed through Tango technology, along with signal-dependent expression of a luciferase reporter gene. When igand binds to MRGPRX2, it activates downstream signaling pathways, leading to the expression of luciferase. Blockade antibodies can inhibit this signal transmission. The luciferase activity measurement indicates the activation level of the signaling pathway and can thus be used to evaluate the in vitro effects of drugs related to MRGPRX2.

Data

Response to Cortistatin-14. The Tango-H_MRGPRX2 CHO-K1 Cell Line (Cat. GM-C37884) at a concentration of 1E4 cells/well (96-well format) was stimulated with serial dilutions of Cortistatin-14 (MCE/HY-P1932) in assay buffer (F12K+1% FBS+1% P.S) for 16 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The maximum induction fold was approximately [8.7]. Data are shown by drug mass concentration.

Response to MrgprX2 anagonis-1. Serial dilutions of the MrgprX2 anagonis-1 (MCE/HY-145191) was incubated with 1E4 cells/well of the Tango-H_MRGPRX2 CHO-K1 Cell Line (Cat. GM-C37884) in a 96-well plate for 1 hour in assay buffer (F12K+1% FBS+1% P.S). Subsequently, the Cortistatin-14 (MCE/HY-P1932) at a concentration of 0.05 μg/well was added, and the coculture proceeded for an additional 16 hours. Firefly luciferase activity was then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated maximum blocking folds of approximately [4.1]. Data are shown by drug molar concentration.

The passage stability of response to Cortistatin-14. The passage 5, 15, and 25 of Tango-H_MRGPRX2 CHO-K1 Cell Line (Cat. GM-C37884) at a concentration of 1E4 cells/well (96-well format) were stimulated with serial dilutions of Cortistatin-14 (MCE/HY-P1932) in assay buffer (F12K+1% FBS+1% P.S) for 16 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). Data are shown by drug mass concentration.

The passage stability of the Tango-H_MRGPRX2 CHO-K1 Cell Line (Cat. GM-C37884) was determined by flow cytometry using APC anti-human MRGX2 Antibody (Biolegend/359006).

Specifications

Cat. No		GM-C37884
Product		Tango-H_MRGPRX2 CHO-K1 Cell Line
Product Format		1 vial of frozen cells
Quantity		5E6 Cells per vail，1 mL
Storage Conditions		Liquid nitrogen immediately upon receipt
Recovery Medium		F12K+10% FBS+1% P.S
Growth medium		F12K+10% FBS+1% P.S+4 μg/mL Blasticidin+100 μg/mL Hygromycin+4 μg/mL Puromycin
Note		None
Freezing Medium		90% FBS+10% DMSO
Growth properties		Adherent
Growth Conditions		37°C, 5% CO₂
Safety considerations		Biosafety Level 2
Mycoplasma Testing		The cell line has been screened to confirm the absence of Mycoplasma species.

Materials

Reagent		Ordering Information
F12K		BOSTER/PYG0036
Fetal Bovine Serum		Cegrogen biotech/A0500-3010
Pen/Strep		Thermo/15140-122
Blasticidin		Genomeditech/GM-040404
Hygromycin		Genomeditech/GM-040403
Puromycin		Genomeditech/GM-040401
Cortistatin-14		MCE/HY-P1932
MrgprX2 antagonist-1		MCE/HY-145191
APC anti-human MRGX2 Antibody		Biolegend/359005
GMOne-Step Luciferase Reporter Gene Assay Kit		Genomeditech/GM-040503

Cell Culture

Cell Recovery

Recovery Medium: F12K+10% FBS+1% P.S

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.

a) Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 - 3 minutes).

b) Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

c) Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium and spin at approximately 176 x g for 5 minutes. Discard supernatant.

d) Resuspend cell pellet with the recommended recovery medium. And dispense into appropriate culture dishes.

e) Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Cell Freezing

Freezing Medium: 90% FBS+10% DMSO

a) Centrifuge at 176 x g for 3 minutes to collect cells.

b) Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.

c) Aliquot 1 mL into each vial.

d) Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.

Cell passage

Growth medium: F12K+10% FBS+1% P.S+4 μg/mL Blasticidin+100 μg/mL Hygromycin+4 μg/mL Puromycin

For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.

a) Remove and discard culture medium.

b) Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.

c) Add 1.0 mL of 0.25% (w/v) Trypsin-EDTA solution to dish and observe cells under an inverted microscope until cell layer is dispersed (usually within 2 to 3 minutes at 37°C).

d) 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.

e) Add 2.0 mL of growth medium to mix well and aspirate cells by gently pipetting.

f) After centrifugation, resuspend the pellet and add appropriate aliquots of the cell suspension to new culture vessels.

g) Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:4 - 1:5 is recommended

Medium Renewal: Every 2 to 3 days

Notes

a) After the stabilization of the cell condition, there will be fewer dead cells post-passage, the cell growth rate will tend to stabilize, cell morphology will become uniform, and the cells will appear robust.

Cell Recovery

Recovery Medium: F12K+10% FBS+1% P.S

c) Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium and spin at approximately 176 x g for 5 minutes. Discard supernatant.

d) Resuspend cell pellet with the recommended recovery medium. And dispense into appropriate culture dishes.

e) Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Cell Freezing

Freezing Medium: 90% FBS+10% DMSO

a) Centrifuge at 176 x g for 3 minutes to collect cells.

b) Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.

c) Aliquot 1 mL into each vial.

d) Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.

Cell passage

Growth medium: F12K+10% FBS+1% P.S+4 μg/mL Blasticidin+100 μg/mL Hygromycin+4 μg/mL Puromycin

For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.

a) Remove and discard culture medium.

b) Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.

c) Add 1.0 mL of 0.25% (w/v) Trypsin-EDTA solution to dish and observe cells under an inverted microscope until cell layer is dispersed (usually within 2 to 3 minutes at 37°C).

e) Add 2.0 mL of growth medium to mix well and aspirate cells by gently pipetting.

f) After centrifugation, resuspend the pellet and add appropriate aliquots of the cell suspension to new culture vessels.

g) Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:4 - 1:5 is recommended

Medium Renewal: Every 2 to 3 days

Notes

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Data

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The passage stability of the Tango-H_MRGPRX2 CHO-K1 Cell Line (Cat. GM-C37884) was determined by flow cytometry using APC anti-human MRGX2 Antibody (Biolegend/359006).

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