KC-2184

293T-CCR4 Cell Line

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Background

CCR4 is a G protein-linked seven transmembrane domain chemokine receptor mainly expressed by T cells, and function as a chemoattractant homing receptor on circulating memory lymphocytes through interaction with its two ligands, CCL17 and CCL22. CCR4 is also widely expressed on malignant T cells and Tregs in the skin and peripheral blood of patients with allergic diseases, and regarded as a potential therapeutic target for cutaneous Tcell lymphoma (CTCL) and atopic dermatitis and bronchial asthma. Its humanized monoclonal antibody, Mogamulizumab, have been approved for treatment CTCL in 2018.

Specifications

Catalog Number:
KC-2184
Cell Line Name:
293T-CCR4 Cell Line
Host Cell Line:
Human HEK293T cell line
Description:
Stable HEK293T cell line expressing exogenous human CCR4 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 CCR4 cell line was generated using a lentiviral vector expressing the human CCR4 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. Yoshie O. CCR4 as a Therapeutic Target for Cancer Immunotherapy. Cancers (Basel). 2021 Nov 4;13(21):5542.
2. Nicolay JP, Albrecht JD, Alberti-Violetti S, Berti E. CCR4 in cutaneous T-cell lymphoma: Therapeutic targeting of a pathogenic driver. Eur J Immunol. 2021 Jul;51(7):1660-1671.
3. Drgona L, Gudiol C, Lanini S, Salzberger B, Ippolito G, Mikulska M. ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Agents targeting lymphoid or myeloid cells surface antigens [II]: CD22, CD30, CD33, CD38, CD40, SLAMF-7 and CCR4). Clin Microbiol Infect. 2018 Jun;24 Suppl 2:S83-S94.
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