KC-2247

K562-OS8-Low-Cell-Line

23678

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Background of K562-OS8-Low-Cell-Line

K562-OS8 stable cell was used as an artificial antigen presenting cells (APCs), OS8 could function as a membrane anchored T cell engager that directly activates TCR in T cell-based assay.

Specifications

Catalog Number	KC-2247
Cell Line Name	K562-OS8-Low-Cell-Line
Host Cell Line	Human K562 cell line
Description	K562 cell line stable expressing membrane OKT3 scFV sequence
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+300µg/mL Hygromycin B
Selection Marker	HygromycinB
Morphology	Lymphoblast
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

K562 OS8 cell line was generated using lentiviral vector expressing a ScFV sequence of anti-human CD3 mAb OKT3 and a C-terminal domain of mouse CD8a which consist of transmembrane and cytoplasmic domains.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 300µg/mL Hygromycin B) 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

atouche, Jean-Baptiste; Sadelain, Michel (2000). "Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells". Nature Biotechnology. 18 (4): 405ÿ409.
Perica, Karlo; Kosmides, Alyssa K; Schneck, Jonathan P (2015). "Linking form to function: Biophysical aspects of artificial antigen presenting cell design". Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1853 (4): 781ÿ790