KC-2435

293T-CD70 Cell Line

24054

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Background of 293T-CD70 Cell Line

CD70, as named as CD27 ligand (CD27L) and TNFSF7, is a type II transmembrane glycoprotein belonging to the TNF superfamily. CD70 is expressed on the surface of DCs, B cells, and T cells and NKs, and it plays an important role in T cell activation after binding with its receptor CD27.

Specifications

Catalog Number	KC-2435
Cell Line Name	293T-CD70 Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CD70 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 1:4-1:5 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 CD70 cell line was generated using a lentiviral vector expressing the CD70 sequence.

Characterization

Figure 1: Characterization of endogenous CD70 expression in 293T cells using FACS.

Figure 2: Characterization of CD70 overexpression in the 293T CD70 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 1:4-1:5 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. Keller, Anna M, Tom A Groothuis, Elise A M Veraar, Marije Marsman, Lucas Maillette de Buy Wenniger, Hans Janssen, Jacques Neefjes, and Jannie Borst. 2007. “Costimulatory Ligand CD70 Is Delivered to the Immunological Synapse by Shared Intracellular Trafficking with MHC Class II Molecules.” Proceedings of the National Academy of Sciences 104 (14): 5989–94.
2. Shaffer, Donald R, Barbara Savoldo, Zhongzhen Yi, Kevin K H Chow, Sunitha Kakarla, David M Spencer, Gianpietro Dotti, et al. 2011. “T Cells Redirected Against CD70 for the Immunotherapy of CD70-Positive Malignancies.” Blood 117 (16). American Society of Hematology: 4304–14.