CD276, also named as human B7 homolog 3 (B7-H3), is a member of the B7 immune protein family. CD276 is expressed on dendritic cells, T cells, B cells, and NK cells, and play an important role immune cell activation as a co stimulatory molecule.
Cell Line Name:
CHO-K1 2Ig B7H3 Cell Line
Host Cell Line:
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% F12K+20% FBS+10% DMSO
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 24 hours
Cell Line Generation
CHOK1 human CD276(B7H3) cell line was generated using a lentiviral vector expressing the human CD276(B7H3) sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (F12K + 10% FBS + 5μg/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 1-2 × 105 cells/mL.
1. Prepare the freezing medium (70% F12K + 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
1. Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, Dong H, Sica GL, Zhu G, Tamada K, Chen L (March 2001). "B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production". Nature Immunology.
2 (3): 269–74 2. Kontos F, Michelakos T, Kurokawa T, Sadagopan A, Schwab JH, Ferrone CR, Ferrone S (October 2020). "B7-H3: an attractive target for antibody-based immunotherapy". Clinical Cancer Research: clincanres.2584.2020
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