KC-5016

293T-mouse-CD8b-Low Cell Line

53998

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Background of 293T-mouse-CD8b-Low Cell Line

CD8B (CD8 Subunit Beta) also known as Ly-3, is a Protein Coding gene. The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The coreceptor CD8 contributes to this antigen-recognition process by binding to a largely invariant region of the MHCI molecule and by promoting intracellular signaling, the effects of which serve to enhance TCR stimuli triggered by cognate ligands. Diseases associated with CD8B include Chromophobe Renal Cell Carcinoma and Uterine Corpus Cancer.

Specifications

Catalog Number	KC-5016
Cell Line Name	293T-mouse-CD8b-Low Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous mouse CD8b gene in Low level
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

Cell Line Generation

293T-mouse-CD8b-Low cell line was generated using a lentiviral vector expressing the mouse CD8b sequence.

Characterization

Figure 1: Characterization of mouse CD8b overexpression in the 293T mouse CD8b stable clone using FACS.

Figure 2: Characterization of mouse CD8b in the 293T mouse CD8b 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. Koh, CH., Lee, S., Kwak, M. et al. CD8 T-cell subsets: heterogeneity, functions, and therapeutic potential. Exp Mol Med 55, 2287–2299 (2023). https://doi.org/10.1038/s12276-023-01105-x.
2. DiSanto JP, Terry LA, Flomenberg N. Generation of anti-human CD8 beta-specific antibodies using transfectants expressing mixed-species CD8 heterodimers. J Immunol Methods. 1991 Jul 26;141(1):123-31. doi: 10.1016/0022-1759(91)90218-5. PMID: 1830894.
3. Pascual-García M, Bértolo C, Nieto JC, Serrat N, Espinosa Í, D'Angelo E, Muñoz R, Rovira R, Vidal S, Prat J. CD8 down-regulation on cytotoxic T lymphocytes of patients with endometrioid endometrial carcinomas. Hum Pathol. 2016 Oct;56:180-8. doi: 10.1016/j.humpath.2016.05.025. Epub 2016 Jun 23. PMID: 27346574.
4. Laugel B, Cole DK, Clement M, Wooldridge L, Price DA, Sewell AK. The multiple roles of the CD8 coreceptor in T cell biology: opportunities for the selective modulation of self-reactive cytotoxic T cells. J Leukoc Biol. 2011 Dec;90(6):1089-99. doi: 10.1189/jlb.0611316. Epub 2011 Sep 27. PMID: 21954283.