Cell Line Information
● Cell lines: KYSE30 (human esophageal squamous cell carcinoma) / Eca-109 (human esophageal cancer)
● Gene name: RBM39
● Cell type: Polyclonal
● Species: Human
● Cell morphology: adherent growth, epithelial-like
● Complete culture medium composition: DMEM + 10% FBS + 1% double antibiotic / RPMI-1640 + 10% FBS + 1% P/S
● Passage ratio: 1:2–1:4
● Passage frequency: Passaged once every 2–3 days.
● Mycoplasma test: Negative

Gene Editing Information

● Construction Method

Gene knockdown refers to a method that reduces or suppresses the expression of a specific gene without permanently disrupting its DNA sequence. Unlike gene knockout, which involves irreversible genetic modifications, gene knockdown achieves its effect by modulating transcriptional or translational processes to decrease the production of the target protein. Gene knockdown is a widely used technique in functional genomics, often employed to investigate the roles of genes within organisms or cells.

Our company employs a CRISPRi system that leverages the properties of the dCas9 protein to inhibit gene transcription by preventing RNA polymerase from binding to the promoter or coding region of a specific gene, without cleaving the DNA.

● Application Scenarios

CRISPRi provides researchers with a powerful tool for precisely and controllably modulating gene expression, offering new solutions for studies in functional genomics, disease modeling, drug screening, and other fields.

● Validate data

Western blot assay results

Cell Culture Instructions

● Cell Revival

1) Remove the cryovial from the liquid nitrogen tank or the −80°C freezer, and promptly place it in a 37°C water bath, gently swirling it to facilitate rapid thawing. To minimize the risk of contamination, you may first transfer the cryovial into a clean, disposable PE glove before immersing it in the water bath.
2) Once the contents of the cryovial have thawed, remove it immediately and wipe the exterior of the cryovial with a 75% ethanol‑soaked gauze.
3) Place the cryovial in a biosafety cabinet, transfer the contents of the cryovial into a centrifuge tube containing approximately 9 mL of complete culture medium, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4) Resuspend the cells in an appropriate volume of complete culture medium, transfer them to a new culture flask, and incubate them in a 5% CO2, 37°C incubator under static conditions.

● Cell passage

When cell density reaches 80% or higher, passaging can be performed at a ratio of 1:2 to 1:4, with passages carried out every 2–3 days. The specific procedure is as follows:
1) Aspirate and discard the culture medium from the flask, add an appropriate volume of PBS, gently swirl to rinse the cells, and then carefully aspirate the PBS.
2) Add approximately 1.0 mL of 0.25% trypsin, gently rock the dish to ensure even coverage of the cells, and then incubate in the cell culture incubator for 2–3 minutes. Under the microscope, observe until the cells become rounded; when about 80% of the cells have detached from the dish wall, add approximately 5 mL of complete culture medium to neutralize the trypsin. Gently pipette up and down to detach the cells from the flask surface, yielding a single-cell suspension. (The volumes specified here are for a T75 flask; adjust accordingly based on your specific experimental conditions.)
3) Transfer the digested cell suspension to a clean 15 mL centrifuge tube, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4) Resuspend the cells in an appropriate volume of complete culture medium, transfer them to new culture flasks at the recommended passage ratio (1:2 to 1:4), and incubate the cells in a 5% CO₂, 37°C incubator under static conditions.

● Cell Cryopreservation

Cell cryopreservation can be performed when cell confluency reaches 80%–90%. The homemade cryopreservation solution consists of DMEM complete medium, 30% FBS, and 10% DMSO. To ensure high post-thaw viability, it is recommended to prepare the cryopreservation solution in advance and pre‑cool it at 4°C.
 

Click the link below to view detailed information.

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