KC-3769

293T-cyno-GPR84-Cell-Line

34744

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Background of 293T-cyno-GPR84-Cell-Line

GPR84 is an inflammation inducing receptor highly expressed on immune cells and is a member of the GPCR superfamily, expected to activate the activity of the urinary angiotensin II receptor. Expected to participate in the neuropeptide signaling pathway. It is a part of the receptor complex.

Specifications

Catalog Number	KC-3769
Cell Line Name	293T-cyno-GPR84-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno-GPR84 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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
In Vivo Validation	NA

Cell Line Generation

293T-cyno-GPR84-cell-line was generated using a lentiviral vector expressing the cyno-GPR84 sequence.

Characterization

Figure 1: Characterization of cyno-GPR84 overexpression in the 293T-cyno-GPR84 stable clone using qPCR.

Figure 2: Characterization of cyno-GPR84 in the 293T-cyno-GPR84 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:4-1:8 every 2-3 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.Wei L, Tokizane K, Konishi H, Yu HR, Kiyama H. Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia. J Neuroinflammation. 2017 Oct 3;14(1):198. doi: 10.1186/s12974-017-0970-y. PMID: 28974234; PMCID: PMC5627487. 2. Aktar R, Rondinelli S, Peiris M. GPR84 in physiology-Many functions in many tissues. Br J Pharmacol. 2024 May;181(10):1524-1535. doi: 10.1111/bph.16206. Epub 2023 Aug 28. PMID: 37533166. 3. Forsman H, Dahlgren C, Mårtensson J, Björkman L, Sundqvist M. Function and regulation of GPR84 in human neutrophils. Br J Pharmacol. 2024 May;181(10):1536-1549. doi: 10.1111/bph.16066. Epub 2023 Mar 27. PMID: 36869866.