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Current position：Product Center > Cell lines > GPCR > AMY：CALCR RAMP > H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line
H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line
DS
Description
Cat. No: GM-C26533
Product: H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line

The Calcitonin Gene Related Peptide (CGRP) family includes several important peptides: Calcitonin (CT), Amylin, and Adrenomedullin (AM). Amylin is produced by the pancreas and is a hormone that regulates nutrient intake. The binding of Amylin to its receptors involves three classes of potential receptors, which are complexes of the Calcitonin Receptor (CALCR) and Receptor Activity Modifying Proteins (RAMPs). RAMPs are a series of type I single-transmembrane proteins that function by forming heterodimers with G protein-coupled receptors. There are three RAMP subtypes: RAMP1, RAMP2, and RAMP3. Although these RAMPs are structurally similar, their amino acid sequences are only about30% identical.

After binding to the receptor, Amylin can form different complexes: CALCR combined with RAMP1 forms the Amy1 complex, CALCR combined with RAMP2 forms the Amy2 complex, and CALCR combined with RAMP3 forms the Amy3 complex.

The H_CALCR RAMP3 (AMY3) Reporter CHO-K1 Cell Line is a clonal, stable cell line that constitutively expresses human CALCR and human RAMP3, along with a signal-dependent expression of a luciferase reporter gene. The binding of Amylin to the receptor complex activates downstream reporter genes, leading to luciferase expression. The luciferase readout indicates the activation level of the signaling pathway and can thus be used to evaluate the in vitro effects of drugs related to the CALCR RAMP3 (AMY3) complex.
Data
Response to Calcitonin salmon. H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line (Cat. GM-C26533) at a concentration of 1.5E4 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 the GMOne-Step Luciferase Reporter Gene Assay Kit(Cat. GM-040503). The maximum induction fold was approximately [31.21]. Data are shown by drug molar concentration.
Response to Amylin. H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line (Cat. GM-C26533) at a concentration of 1E4 cells/well (96-well format) was stimulated with serial dilutions of Amylin (Genscript/RP11278CN) in assay buffer (F12K +1% FBS+1% P.S) for 6 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 [43.5]. Data are shown by drug molar concentration.
The passage stability of response to Amylin. The passage 7, 12, 17, 22, and 27 of H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line (Cat. GM-C26533) at a concentration of 1E4 cells/well (96-well format) was stimulated with serial dilutions of Amylin (Genscript/RP11278CN) in assay buffer (F12K +1% FBS+1% P.S) for 6 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). Data are shown by drug molar concentration.
The mRNA expression levels of H_CALCR in the H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line(Cat. GM-C26533) were determined by RT-qPCR.
The mRNA expression levels of H_RAMP3 in the H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line(Cat. GM-C26533) were determined by RT-qPCR.
Specifications
Cat. NoGM-C26533
ProductH_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line
Product Format1 vial of frozen cells
Quantity5E6 Cells per vial,1 mL
Storage ConditionsLiquid nitrogen immediately upon receipt
Recovery MediumF12K +10% FBS+1% P.S
Growth mediumF12K+10% FBS+1% P.S+4 μg/mL Blasticidin+200 μg/mL G418+4 μg/mL Puromycin
NoteNone
Freezing Medium90% FBS+10% DMSO
Growth propertiesAdherent
Growth Conditions37°C, 5% CO₂
Safety considerationsBiosafety Level 2
Mycoplasma TestingThe cell line has been screened to confirm the absence of Mycoplasma species.
Materials
ReagentOrdering Information
F12KBOSTER/PYG0036
Fetal Bovine SerumCegrogen biotech/A0500-3010
Pen/StrepThermo/15140-122
BlasticidinGenomeditech/GM-040404
G418Genomeditech/GM-040402
PuromycinGenomeditech/GM-040401
Calcitonin salmon (Salmon calcitonin)GlpBio/GC32851
Amylin, human, amideGenScript/RP11278CN
GMOne-Step Luciferase Reporter Gene Assay KitGenomeditech/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+200 μg/mL G418+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.


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Current Position：Product Center > Cell lines > GPCR > AMY：CALCR RAMP > H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line
classify
H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line
DS
Description
Cat. No: GM-C26533
Product: H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line

The Calcitonin Gene Related Peptide (CGRP) family includes several important peptides: Calcitonin (CT), Amylin, and Adrenomedullin (AM). Amylin is produced by the pancreas and is a hormone that regulates nutrient intake. The binding of Amylin to its receptors involves three classes of potential receptors, which are complexes of the Calcitonin Receptor (CALCR) and Receptor Activity Modifying Proteins (RAMPs). RAMPs are a series of type I single-transmembrane proteins that function by forming heterodimers with G protein-coupled receptors. There are three RAMP subtypes: RAMP1, RAMP2, and RAMP3. Although these RAMPs are structurally similar, their amino acid sequences are only about30% identical.

After binding to the receptor, Amylin can form different complexes: CALCR combined with RAMP1 forms the Amy1 complex, CALCR combined with RAMP2 forms the Amy2 complex, and CALCR combined with RAMP3 forms the Amy3 complex.

The H_CALCR RAMP3 (AMY3) Reporter CHO-K1 Cell Line is a clonal, stable cell line that constitutively expresses human CALCR and human RAMP3, along with a signal-dependent expression of a luciferase reporter gene. The binding of Amylin to the receptor complex activates downstream reporter genes, leading to luciferase expression. The luciferase readout indicates the activation level of the signaling pathway and can thus be used to evaluate the in vitro effects of drugs related to the CALCR RAMP3 (AMY3) complex.
Data
Response to Calcitonin salmon. H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line (Cat. GM-C26533) at a concentration of 1.5E4 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 the GMOne-Step Luciferase Reporter Gene Assay Kit(Cat. GM-040503). The maximum induction fold was approximately [31.21]. Data are shown by drug molar concentration.
Response to Amylin. H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line (Cat. GM-C26533) at a concentration of 1E4 cells/well (96-well format) was stimulated with serial dilutions of Amylin (Genscript/RP11278CN) in assay buffer (F12K +1% FBS+1% P.S) for 6 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 [43.5]. Data are shown by drug molar concentration.
The passage stability of response to Amylin. The passage 7, 12, 17, 22, and 27 of H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line (Cat. GM-C26533) at a concentration of 1E4 cells/well (96-well format) was stimulated with serial dilutions of Amylin (Genscript/RP11278CN) in assay buffer (F12K +1% FBS+1% P.S) for 6 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). Data are shown by drug molar concentration.
The mRNA expression levels of H_CALCR in the H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line(Cat. GM-C26533) were determined by RT-qPCR.
The mRNA expression levels of H_RAMP3 in the H_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line(Cat. GM-C26533) were determined by RT-qPCR.
Specifications
Cat. NoGM-C26533
ProductH_CALCR RAMP3(AMY3) Reporter CHO-K1 Cell Line
Product Format1 vial of frozen cells
Quantity5E6 Cells per vial,1 mL
Storage ConditionsLiquid nitrogen immediately upon receipt
Recovery MediumF12K +10% FBS+1% P.S
Growth mediumF12K+10% FBS+1% P.S+4 μg/mL Blasticidin+200 μg/mL G418+4 μg/mL Puromycin
NoteNone
Freezing Medium90% FBS+10% DMSO
Growth propertiesAdherent
Growth Conditions37°C, 5% CO₂
Safety considerationsBiosafety Level 2
Mycoplasma TestingThe cell line has been screened to confirm the absence of Mycoplasma species.
Materials
ReagentOrdering Information
F12KBOSTER/PYG0036
Fetal Bovine SerumCegrogen biotech/A0500-3010
Pen/StrepThermo/15140-122
BlasticidinGenomeditech/GM-040404
G418Genomeditech/GM-040402
PuromycinGenomeditech/GM-040401
Calcitonin salmon (Salmon calcitonin)GlpBio/GC32851
Amylin, human, amideGenScript/RP11278CN
GMOne-Step Luciferase Reporter Gene Assay KitGenomeditech/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+200 μg/mL G418+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.


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