KC-1153

CHOK1-CD16a-V158 Cell Line

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Background of CHOK1-CD16a-V158 Cell Line

CD16, also named FC gamma RIII, is a low or intermediate affinity FC receptor, and has been identified as two receptors including FcγRIIIa (CD16a) and FcγRIIIb (CD16b). It is involved in phagocytosis, secretion of enzymes and inflammatory mediators, antibody-dependent cytotoxicity and clearance of immune complexes.

Specifications

Catalog Number	KC-1153
Cell Line Name	CHOK1-CD16a-V158 Cell Line
Clone Number	NA
Host Cell Line	CHOK1
Description	CHOK1 cell line stable expressing human FCGR3A amino acid bearing V158 mutation
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 + 5μg/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 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1-CD16a-V158 Cell Line was generated using lentiviral vector expressing human CD16a sequence bearing V158 mutation.

Characterization

Figure 1: Characterization of CD16a overexpressing in CHO-K1 stable clones using FACS.

Cell Resuscitation

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 1-3 × 10⁵ cells/mL.

Cell Freezing

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.

References

1. Tada, Minoru, Akiko Ishii-Watabe, Takuo Suzuki, and Nana Kawasaki. 2014. Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies. Edited by Paul Zhou. PLoS ONE 9 (4): e95787–89. doi:10.1371/journal.pone.0095787.
2. Koene, H R, M Kleijer, J Algra, D Roos, A E von dem Borne, and M de Haas. 1997. Fc gammaRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell Fc gammaRIIIa, Independently of the Fc gammaRIIIa-48L/R/H Phenotype. Blood 90 (3): 1109–14.