KC-3703

MDA-MB-231-STING-KO-2A2-Cell-Line

Stable MDA-MB-231 clone with human STING gene knockout, No.2A2

40702

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Background of MDA-MB-231-STING-KO-2A2-Cell-Line

STING encodes a five transmembrane protein that functions as a major regulator of the innate immune response to viral and bacterial infections. It is the facilitator of innate immune signaling that acts as a sensor of cytosolic DNA from bacteria and viruses and promotes low production of type I interferon. STING acts by binding cyclic dinucleotides: recognizes and binds cyclic di-GMP (c-di-GMP), a second messenger produced by bacteria, and cyclic GMP-AMP (cGAMP), a messenger produced by cGAS in response to DNA virus in the cytosol.

Specifications

Catalog Number	KC-3703
Cell Line Name	MDA-MB-231-STING-KO-2A2-Cell-Line
Host Cell Line	MDA-MB-231
Description	Stable MDA-MB-231 clone with human STING gene knockout, No.2A2
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	N/A
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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 38 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

MDA-MB-231-STING-KO-2A2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of MDA-MB-231-STING-KO-2A2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of MDA-MB-231-STING-KO-2A2 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of MDA-MB-231-STING-KO-2A2 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS)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 1:3-1:6 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

Samson N, Ablasser A. The cGAS-STING pathway and cancer. Nat Cancer. 2022 Dec;3(12):1452-1463. doi: 10.1038/s43018-022-00468-w. Epub 2022 Dec 12. PMID: 36510011.
Xie J, Li Y, Shen X, Goh G, Zhu Y, Cui J, Wang LF, Shi ZL, Zhou P. Dampened STING-Dependent Interferon Activation in Bats. Cell Host Microbe. 2018 Mar 14;23(3):297-301.e4. doi: 10.1016/j.chom.2018.01.006. Epub 2018 Feb 22. PMID: 29478775; PMCID: PMC7104992.
Zhang R, Qin X, Yang Y, Zhu X, Zhao S, Zhang Z, Su Q, Zhao Z, Yin X, Meng X, Zhang Z, Li Y. STING1 is essential for an RNA-virus triggered autophagy. Autophagy. 2022 Apr;18(4):816-828. doi: 10.1080/15548627.2021.1959086. Epub 2021 Aug 2. PMID: 34338134; PMCID: PMC9037512.