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Figure 1. Calcium flux in α1B–expressing Chem-1 cell line induced by Epinephrine. α1B expressing Chem-1 cells and Wild-Type Chem-1 cells were loaded ...
ChemiSCREEN™ Human Recombinant α1B Adrenergic Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant α1B Adrenergic Receptor Calcium-Optimized Stable Cell Line
Product Overview:
Full-length human ADRA1B cDNA encoding α1B
Background Information:
The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenoceptors (Bylund et al., 1994). The three members of the α1 subclass of adrenoceptors, α1A, α1B and α1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue. Overexpression of a constitutively active α1B mutant in the heart of transgenic mice resulted in cardiac hypertrophy with increased heart weight/body weight ratios. Analysis of α1B knock out mice has provided evidence that α1B is a mediator of blood pressure and aortic contractile responses induced by α1 agonists (Milano et al., 1994). The locomotor and rewarding effects of pysochostimulants and opiates were suppressed in mice lacking α1B-adrenergic receptors (Drouin et al. 2002). Millipore’s cloned human α1B-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant α1B expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between α1B and its ligands.
Application Notes:
Calcium flux assay, ligand binding assays
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Host Cells:
Chem-1, an adherent cell line expressing the endogenous promiscuous G-protein, Gα15.
Packaging:
2 x 106 cells/vial
Materials Required but Not Delivered:
Chem-1 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020-A)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250g/mL Genetecin/G-418
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-1 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020-A)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250g/mL Genetecin/G-418
Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
Chem-1 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)
Gene Symbol:
- ADRA1B
- ALPHA1BAR
- ADRA1
Presentation:
Cells are frozen at 2 x 106 cells/mL in 90% fetal bovine serum/10% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.
Quality Assurance:
EC50 for calcium mobilization by Epinephrine: ~1.7 nM
Storage Conditions:
1.Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years.
2.Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37ºC with 5% CO2.
3.After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4.When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.
5.Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6.Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.
7.Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.
2.Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37ºC with 5% CO2.
3.After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.
4.When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca++ and Mg++ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.
5.Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.
6.Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 106 cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 106 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.
7.Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.