KC-4445

MC38-CCR8-Low Cell Line

49113

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Background of MC38-CCR8-Low Cell Line

CCR8, This gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. Chemokines and their receptors are important for the migration of various cell types into the inflammatory sites. This receptor protein preferentially expresses in the thymus. I-309, thymus activation-regulated cytokine (TARC) and macrophage inflammatory protein-1 beta (MIP-1 beta) have been identified as ligands of this receptor. Studies of this receptor and its ligands suggested its role in regulation of monocyte chemotaxis and thymic cell apoptosis. More specifically, this receptor may contribute to the proper positioning of activated T cells within the antigenic challenge sites and specialized areas of lymphoid tissues. This gene is located at the chemokine receptor gene cluster region.

Specifications

Catalog Number	KC-4445
Cell Line Name	MC38-CCR8-Low Cell Line
Clone Number	11#
Host Cell Line	MC38
Description	MC38 cell line stably expressing exogenous CCR8 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 5 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:6 every 2-3 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

MC38-CCR8-Low cell line was generated using a lentiviral vector expressing the CCR8 sequence.

Characterization

Figure1: Characterization of CCR8 overexpression in MC38-CCR8-Low stable clone using FACS.

Figure2: Characterization of MC38-CCR8-Low cell line stable clone using PCR sequencing.

Cell Resuscitation

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

Cell Freezing

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

References

1.Peng Q, Jiang H, Cheng X, Wang N, Zhou S, Zhang Y, Yang T, Chen Y, Zhang W, Lv S, Nan W, Wang J, Fan GH, Li J, Zhang J. Cryo-EM Structure and Biochemical Analysis of the Human Chemokine Receptor CCR8. Biochemistry. 2024 Aug 6;63(15):1892-1900. doi: 10.1021/acs.biochem.4c00121. Epub 2024 Jul 10. PMID: 38985857.
2.Napolitano M, Zingoni A, Bernardini G, Spinetti G, Nista A, Storlazzi CT, Rocchi M, Santoni A. Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues. J Immunol. 1996 Oct 1;157(7):2759-63. PMID: 8816377.
3.Tiffany HL, Lautens LL, Gao JL, Pease J, Locati M, Combadiere C, Modi W, Bonner TI, Murphy PM. Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309. J Exp Med. 1997 Jul 7;186(1):165-70. doi: 10.1084/jem.186.1.165. PMID: 9207005; PMCID: PMC2198957.