KC-1755

H22-mouse-CD274-KO-3A3-Cell-Line

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Background of H22-mouse-CD274-KO-3A3-Cell-Line

CD27, also named as TNFRSF7, is a transmembrane protein belonging to the TNF receptor superfamily, and not only functioned as a co-stimulatory immune checkpoint molecule in T cell activation, but also play a critical role in regulating B-cell activation and immunoglobulin synthesis after binding with its ligand, CD70. CD27 is also promising immunotherapy target for cancer treatment, its monoclonal antibody Varlilumab are in phase I for solid tumor and hematologic malignancies.

Specifications

Catalog Number	KC-1755
Cell Line Name	H22-mouse-CD274-KO-3A3-Cell-Line
Host Cell Line	Mouse H22 cell line
Description	H22 cell line stable clone with mouse CD274 gene knockout
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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS
Morphology	Round
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
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

H22 mouse-CD274 KO cell line was generated using the CRISPR method.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI-1640 supplemented with 10% FBS)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% RPMI-1640 + 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

Lens SM, de Jong R, Hintzen RQ, Koopman G, van Lier RA, van Oers RH (Jun 1995). "CD27-CD70 interaction: unravelling its implication in normal and neoplastic B-cell growth". Leukemia & Lymphoma. 18 (1ÿ2): 51ÿ9.