KC-5293

A549-CD69 Cell Line

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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	One vial 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.

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.