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KC-5892

Jurkat-NFAT-Luc2-CD3E-KO-CD16a-V158 Cell Line

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Home » Jurkat-NFAT-Luc2-CD3E-KO-CD16a-V158 Cell Line

Background of Jurkat-NFAT-Luc2-CD3E-KO-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. NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. Nuclear factor of activated T cells (NFAT), which is the pharmacological target of immunosuppressants cyclosporine and tacrolimus, has been shown to play an important role not only in T cells (immune system), from which their ame is derived, but also in many biological events. The activity of NFAT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a primary target for inhibition by cyclosporin A and FK506. Calcineurin controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells by interacting with an N-terminal regulatory domain conserved in the NFAT family. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability to bind to certain regulatory elements in cytokine genes. NFAT is also notable for its ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun) family to composite NFAT: AP-1 sites, found in the regulatory regions of many genes that are inducibly transcribed by immune-system cells.

Specifications

Catalog NumberKC-5892
Cell Line NameJurkat-NFAT-Luc2-CD3E-KO-CD16a-V158 Cell Line
NCBI/UniProt Accession NumberNM_000569.8
Clone Number22#
Host Cell LineJurkat-NFAT-Luc2-CD3E-KO
DescriptionJurkat cell line stable expressing exogenous luciferase gene under the control of NFAT responsive element and human CD3E gene knockout with human CD16a amino acid bearing F158V mutation
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640 + 10% FBS + 1μg/mL Puromycin
Selection MarkerPuromycin
MorphologySuspension
SubcultureSplit saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative

Cell Line Generation

The Jurkat NFAT-Luc2-CD3E-KO-CD16a-F158 cell line stably expresses an exogenous luciferase gene under the control of the NFAT-responsive element, with knockout of the human CD3E genes and the F158V mutation of the human CD16a gene.

Characterization

Figure 1: Characterization of CD16a-V158 overexpression in Jurkat-NFAT-Luc2-CD3E-KO-CD16a-V158 reporter cell line using FACS.

Figure 2: Jurkat NFAT-Luc2-CD3E-KO-CD16a-V158 cells and Raji cells were seeded into 96-well plates, treated with Rituximab for 6 hours, and then read out using Bright-Glo Detection System.

Figure3: Characterization of Jurkat NFAT-Luc2-CD3E-KO-CD16a-V158 cell line stable clone using PCR sequencing.

Cell Resuscitation

  1. Prewarm culture medium (RPMI1640 + 10% FBS + 1μg/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 1:4-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% RPMI1640 + 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. ¡ùDevelopment of a Cell-Based Assay Measuring the Activation of FcÎÛRIIa for the Characterization of Therapeutic Monoclonal Antibodies.¡ì Edited by Paul Zhou. PLoS ONE 9 (4): e95787ÿ89. 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. ¡ùFc gammaRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell Fc gammaRIIIa, Independently of the Fc gammaRIIIa-48L/R/H Phenotype..¡ì Blood 90 (3): 1109ÿ14.

Use License Agreement

Research Use Only.
Not for use in diagnostic procedures or therapeutic applications.
Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.

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