KC-2779

Calu6 B7H3 KO 3A3 Cell Line

27167

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Background of Calu6 B7H3 KO 3A3 Cell Line

B7-H3 is a 316 amino acid-long type I transmembrane protein, existing in two isoforms determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains. B7-H3 mRNA is expressed in most normal tissues. In contrast, B7-H3 protein has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. However, B7-H3 protein is expressed at high frequency on many different cancer types (60% of all cancers).

Specifications

Catalog Number	KC-2779
Cell Line Name	Calu6 B7H3 KO 3A3 Cell Line
Host Cell Line	Calu6
Description	Stable Calu6 clone with human B7H3 gene knockout, No.3A3
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% MEM + 0.01Mm NEAA+20% FBS + 10% DMSO
Propagation Medium	MEM + 0.01Mm NEAA + 10% FBS
Selection Marker	N/A
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 48 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Calu-6 -B7H3 knockout cell line was generated using a CRISPR method.

Characterization

Figure 1: Characterization of B7H3 knockout in Calu-6 using PCR sequencing.

Figure 2: Characterization of B7H3 knockout in Calu-6 using FACS.

Cell Resuscitation

Prewarm culture medium (MEM + 0.01Mm NEAA + 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% MEM + 0.01Mm NEAA+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

Zhang GB, Chen YJ, Shi Q, Ma HB, Ge Y, Wang Q, Jiang Z, Xu Y, Zhang XG (June 2004). Human recombinant B7-H3 expressed in E. coli enhances T lymphocyte proliferation and IL-10 secretion in vitro. Acta Biochimica et Biophysica Sinica. 36 (6): 430–6. doi:10.1093/abbs/36.6.430. PMID 15188059.