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Calcium flux in CXCR1-expressing Chem-1 cell line induced by IL-8 and GROα. CXCR1-expressing Chem-1 cells and wild-type Chem-1 cells (Chemicon catalo ...
ChemiSCREEN™ Human Recombinant CXCR1 Chemokine Receptor Calcium-Optimized Stable Cell Line
Description:
ChemiSCREEN™ Human Recombinant CXCR1 Chemokine Receptor Calcium-Optimized Stable Cell Line
Trade Name:
- ChemiScreen
- Chemicon (Millipore)
Product Overview:
Full-length human CXCR1 cDNA
Background Information:
CXCR1 (also known as IL-8RA) is a Gi/o-coupled GPCR expressed on neutrophils, CD8(+) T cells, and intestinal epithelial cells (Murphy, 1997; Sturm et al., 2005; Takata et al., 2004). CXCR1 binds with high affinity to the chemokine interleukin-8 (IL-8) and much lower affinity to GROα, whereas a related receptor, CXCR2, binds with equal affinity to IL-8 and GROα (Hall et al., 1999). CXCR1 has been proposed to be the primary regulator of neutrophil response to IL-8 in sepsis (Cummings et al., 1999). Antagonists of CXCR1 and CXCR2 have been developed and display efficacy in animal models of reperfusion injury (Bertini, et al., 2004; Kaneider et al., 2005). Chemicon's cloned human CXCR1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant CXCR1 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between CXCR1 and its ligands.
Application Notes:
Calcium flux assay, ligand binding assays
Species Reactivity:
Human
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.
Descriptive Text:
Concentration:
2x106cells/vial
Host Cells:
Chem-1, an adherent cell line expressing the promiscuous G-protein, Gα15 and lacking endogenous expression of CXCR1 or CXCR2.
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)
250µg/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:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
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
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:
DMEM with 4.5 g/L glucose and 4 mM glutamine
20% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep
10% DMSO (cell culture grade)
Packaging:
2x106cells/vial
Presentation:
Cells are frozen at 2 x 106 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma
Note: This CXCR1 cell line in the Chem-1 background replaces the CXCR1-Chem-3 cell line previously offered under this catalog number
Note: This CXCR1 cell line in the Chem-1 background replaces the CXCR1-Chem-3 cell line previously offered under this catalog number
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.
Cell Line Type:
GPCR Cell Lines
Cell Type:
Chem-1
Gene Symbol:
- IL8RA
- CMKAR1
- CKR-1
- C-C
- C-C-CKR-1
- CXCR-1
- IL8RBA
- IL8R1
- CDw128a
- CD128
- CXCR1
- CD181
Format:
Cell Line
Quality Assurance:
SPECIFICATIONS: EC50 for calcium mobilization by IL-8: ~ 4 nM
EC50 for calcium mobilization by GROα: >200 nM
Signal/noise ratio at IL-8 Emax: 603
EC50 for calcium mobilization by GROα: >200 nM
Signal/noise ratio at IL-8 Emax: 603
Protein or Enzyme Type:
GPCRs
Species:
Human