H_CALCR β-Arrestin Reporter CHO-K1 Cell Line

Cat. No: GM-C42212
Product: H_CALCR β-Arrestin Reporter CHO-K1 Cell Line

CALCR(Calcitonin Receptor) is a G protein-coupled receptor encoded by the CALCR gene, mainly found in bones, kidneys, and the central nervous system. It regulates calcium metabolism and bone homeostasis by inhibiting osteoclast activity and promoting calcium excretion, playing a significant role in conditions like osteoporosis.

When calcitonin binds to CALCR, it activates different signaling pathways through G protein coupling mechanisms. Activates Gs protein, increasing adenylate cyclase activity and cAMP levels, which inhibits osteoclasts and reduces bone resorption. Stimulates phospholipase C to release intracellular calcium, enhancing physiological responses and regulating bone metabolism and inflammation. In summary, CALCR is vital for calcium homeostasis and may be a therapeutic target for related diseases.

H_CALCR β-Arrestin Reporter CHO-K1 Cell Line is a clonal stable CHO-K1 cell line constructed using lentiviral technology, constitutive expression of the CALCR gene, and detected using enzyme fragment complementation (EFC) technology. When the ligand binds to CALCR, it recruits β-arrestin fused with a downstream luciferase reporter gene. Upon addition of the luciferase substrate, the enzyme catalyzes the substrate reaction, producing a detectable luminescent signal. Therefore, this system can be used to evaluate the in vitro efficacy of related drugs.

Data

Response to Calcitonin salmon. The H_CALCR β-Arrestin Reporter CHO-K1 Cell Line (Cat. GM-C42212) at a concentration of 1E4 cells/well (96-well format) was stimulated with serial dilutions of Calcitonin salmon (GLPBIO/GC32851) in assay buffer (F12K+1% FBS+1% P.S) for 6 hours. The firefly luciferase activity was measured using a detection kit. The maximum induction fold was approximately [31.5]. Data are shown by drug molar concentration.

Specifications

Cat. No		GM-C42212
Product		H_CALCR β-Arrestin Reporter CHO-K1 Cell Line
Product Format		1 vial of frozen cells
Quantity		5E6 Cells per vial,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 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		ExCell/FSP500
Pen/Strep		Thermo/15140-122
Puromycin		Genomeditech/GM-040401
Calcitonin salmon (Salmon calcitonin)		GlpBio/GC32851

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 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 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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