KC-2034
293T-human-SEMA4D-Cell-Line
Background of 293T-human-SEMA4D-Cell-Line
SEMA4D is a signaling protein. It plays an important role in T cell initiation, positive regulation of phosphatidylinositol 3-kinase signaling, regulation of neuronal projection development, and regulation of phosphate metabolism. SEMA4D is highly expressed in fibrotic liver, and knocking out SEMA4D can reduce liver fibrosis. Therefore, targeting SEMA4D may be a potential treatment for liver fibrosis.
Specifications
| Catalog Number | KC-2034 |
|---|---|
| Cell Line Name | 293T-human-SEMA4D-Cell-Line |
| Host Cell Line | 293T |
| Description | Stable HEK293T clone expressing exogenous human SEMA4D 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 human SEMA4D cell line was generated using a lentiviral vector expressing the human SEMA4D sequence.
Characterization
Figure 1: Characterization of human SEMA4D overexpression in the 293T human SEMA4D stable clone using FACS.
Cell Resuscitation
- Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 9.0mL 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 1:4-1:8 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
- Wang L, Li D, Zhu Z, Liao Y, Wu J, Liu Y, Yang R, Dai H, Wu Z, Sun X. Knockout of Sema4D alleviates liver fibrosis by suppressing AOX1 expression. Pharmacol Res. 2023 Sep;195:106886. doi: 10.1016/j.phrs.2023.106886. Epub 2023 Aug 15. PMID: 37591326.
- Wu JH, Li YN, Chen AQ, Hong CD, Zhang CL, Wang HL, Zhou YF, Li PC, Wang Y, Mao L, Xia YP, He QW, Jin HJ, Yue ZY, Hu B. Inhibition of Sema4D/PlexinB1 signaling alleviates vascular dysfunction in diabetic retinopathy. EMBO Mol Med. 2020 Feb 7;12(2):e10154. doi: 10.15252/emmm.201810154. Epub 2020 Jan 13. PMID: 31943789; PMCID: PMC7005627.
- Ishii T, Ruiz-Torruella M, Kim JY, Kanzaki H, Albassam A, Wisitrasameewong W, Shindo S, Pierrelus R, Heidari A, Kandalam U, Nakamura S, Movila A, Minond D, Kawai T. Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis. J Cell Mol Med. 2023 Jun;27(12):1750-1756. doi: 10.1111/jcmm.17416. Epub 2023 May 11. PMID: 37170687; PMCID: PMC10273054.