KC-5293

A549-CD69 Cell Line

60656

Home » A549-CD69 Cell Line

Background of A549-CD69 Cell Line

This gene encodes a member of the calcium dependent lectin superfamily of type II transmembrane receptors. Expression of the encoded protein is induced upon activation of T lymphocytes, and may play a role in proliferation. Furthermore, the protein may act to transmit signals in natural killer cells and platelets.

Specifications

Catalog Number	KC-5293
Cell Line Name	A549-CD69 Cell Line
NCBI/UniProt Accession Number	NM_001781
Clone Number	3-5#
Host Cell Line	A549
Description	Stable A549 clone expressing exogenous CD69 gene
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 + 1μg/mL Puromycin
Selection Marker	Puromycin
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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

A549-CD69 cell line was generated using a lentiviral vector expressing the CD69 sequence.

Characterization

Figure 1: Characterization of CD69 overexpression in the A549-CD69 stable clone using FACS.

Figure 2: Characterization of CD69 in the A549-CD69 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 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 1:4-1:5 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. Ishikawa C, Kawakami H, Uchihara JN, Senba M, Mori N. CD69 overexpression by human T-cell leukemia virus type 1 Tax transactivation. Biochim Biophys Acta. 2013 Jun;1833(6):1542-52. doi: 10.1016/j.bbamcr.2013.03.006. Epub 2013 Mar 16. PMID: 23507197.
2. Montraveta A, Lee-Vergés E, Roldán J, Jiménez L, Cabezas S, Clot G, Pinyol M, Xargay-Torrent S, Rosich L, Arimany-Nardí C, Aymerich M, Villamor N, López-Guillermo A, Pérez-Galán P, Roué G, Pastor-Anglada M, Campo E, López-Guerra M, Colomer D. CD69 expression potentially predicts response to bendamustine and its modulation by ibrutinib or idelalisib enhances cytotoxic effect in chronic lymphocytic leukemia. Oncotarget. 2016 Feb 2;7(5):5507-20. doi: 10.18632/oncotarget.6685. PMID: 26701728; PMCID: PMC4868702.