Content Loading...
Content Loading...
Product Images
Figure 1. Calcium flux in P2Y4–expressing 1321N1 cell line induced by UTP, ATP, ADP, and Benzoylbenzoyl-ATP (Bz-ATP). P2Y4- expressing 1321N1 cells ...
ChemiSCREEN™ Human Recombinant P2Y4 Purinergic Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant P2Y4 Purinergic Receptor Calcium-Optimized Stable Cell Line
Trade Name:
Chemicon (Millipore)
Qty/Pk:
2 vials
Product Overview:
Proprietary plasmid containing full-length human P2Y4 cDNA encoding P2Y4 . The stable clonal cell line was selected by resistance to geneticin, followed by limited dilution cloning. The cell line was tested and found to have equivalent EC50 and signal at 1, 3 and 6 weeks of continuous culture.
Background Information:
The P2Y GPCRs serve as receptors for extracellular nucleotides. The P2Y4 receptor is activated by UTP, and also by ATP in rodents, and couples to Gq to increase intracellular calcium (von Kügelgen, 2006). P2Y4 is expressed prominently in the intestinal epithelium, where it regulates intestinal chloride secretion. P2Y4 therefore is likely to be involved in infectious diarrhea caused by nucleotides released upon infection with enteropathogenic bacteria (Robaye et al., 2003). Millipore’s cloned human P2Y4 -expressing cell line is made in the 1321N1host, which supports high levels of recombinant P2Y4 expression on the cell surface. Thus, the cell line is an ideal tool for screening for agonists and antagonists of P2Y4 .
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.
Species:
Human
Quality Assurance:
Table I. Comparison of EC50 values of P2Y4-expressing 1321N1 cells with values described in the literature.
| ligand | assay | potency | Efficacy (% of maximum) | Reference |
|---|---|---|---|---|
| UTP | Calcium | EC50 = 73.2 nM | 100% | Figure 1 |
| UTP | Inositol phosphate | EC50 = 300-2400 nM | 100% | Communi et al., 1995, 1996 |
| UTP | Calcium | EC50 = 530 nM | 100% | Herold et al., 2004 |
| ATP | Calcium | EC50 = 2.5 μM | 42% | Figure 1 |
| ATP | Inositol phosphate | EC50 = 43 μM | 35% | Communi et al., 1995, 1996 |
| ADP | Calcium | EEC50 = 10 μM | 0% | Figure 1 |
| ADP | Inositol phosphate | "barely detectable" | "barely detectable" | Communi et al., 1995 |
Brand Family:
Chemicon
Host Cells:
1321N1
Presentation:
Cells are frozen at 2 x 106 cells/mL in 90% FBS/10% DMSO. Cell line tests negative for mycoplasma.
Protein Target:
P2Y4
Ligand Type:
non-peptide
Storage Conditions:
1.Immediately upon receipt, thaw cells or place cells in liquid nitrogen.
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 1321N1 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 1321N1 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 1321N1 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 1321N1 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 1321N1 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 1321N1 Plating Media for plating for calcium assay.
Target Sub-Family:
P2Y / Purinergic (metabotropic)
Promotional Text:
For special offers Click here http://www.millipore.com/drugdiscovery/dd3/gpcrtargetsolutions
GPCR Class:
A
Packaging:
2 x 106 cells/vial
Product Name:
ChemiSCREEN™ Human Recombinant P2Y4 Purinergic Receptor Calcium-Optimized Stable Cell Line
Entrez Gene Number:
Materials Required but Not Delivered:
1321N1 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)
250μg/mL Genetecin/G-418
1321N1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
1321N1 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)
250μg/mL Genetecin/G-418
1321N1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
1321N1 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)