KC-2494

293T-CCR2b-High Cell Line

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Background

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to Gi and Gq family members. CCR2 has been found to be expressed by multiple cell types including monocytes, dendritic cells (DCs), endothelial cells, certain T cell subsets, and cancer cells. CCR2 mediates agonistdependent calcium mobilization and inhibition of adenylyl cyclase, and it can also be a coreceptor with CD4 for HIV1 infection. CCL2-CCR2 signaling axis is implicated in many inflammatory and neurodegenerative diseases such atherosclerosis, multiple sclerosis, asthma, neuropathic pain, diabetic nephropathy, and cancer. PF-4136309 is an inhibitor of CCR3.

Specifications

Catalog Number:
KC-2494
Cell Line Name:
293T-CCR2b-High Cell Line
Host Cell Line:
Human HEK293T cell line
Description:
Stable HEK293T cell line expressing exogenous human CCR2b gene
Quantity:
One vial of frozen cells (5×106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% DMEM+20% FBS+10% DMSO
Propagation Medium:
DMEM+10%FBS+1μg/mL Puromycin
Selection Marker:
Puromycin
Morphology:
Epithelial
Subculture:
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL
Incubation:
37 °C with 5% CO2
Storage:
Liquid nitrogen immediately upon receiving
Doubling Time:
Approximately 30 hours
Mycoplasma Status:
Negative

Cell Line Generation

293T human CCR2b cell line was generated using a lentiviral vector expressing the human CCR2b sequence.

Characterization

Application

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1μg/mL Puromycin) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage

References

1. Minsaas L, Planagum½Ç J, Madziva M, Krakstad BF, Masi½Ç-Balagu¹È M, Katz AA, Aragay AM. Filamin a binds to CCR2B and regulates its internalization. PLoS One. 2010 Aug 17;5(8):e12212. doi: 10.1371/journal.pone.0012212. PMID: 20808917; PMCID: PMC2923182.
2. Markx D, Schuhholz J, Abadier M, Beier S, Lang M, Moepps B. Arginine 313 of the putative 8th helix mediates GÏ«q/14 coupling of human CC chemokine receptors CCR2a and CCR2b. Cell Signal. 2019 Jan;53:170-183. doi: 10.1016/j.cellsig.2018.10.007. Epub 2018 Oct 12. PMID: 30321592.
3. Hao Q, Vadgama JV, Wang P. CCL2/CCR2 signaling in cancer pathogenesis. Cell Commun Signal. 2020 May 29;18(1):82. doi: 10.1186/s12964-020-00589-8. PMID: 32471499; PMCID: PMC7257159.
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