HEK293-CTLA4

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The HEK293-CTLA4 cell line is a stable recombinant HEK293 cell line engineered to express the CTLA4 receptor at a medium-low level, approximately 4,000 molecules per cell. This cell line was developed using inscreenex's landing pad technology, ensuring precise and reproducible integration of the CTLA4 gene at a specific, pre-validated genomic locus. CTLA4, also known as CD152, is an immune checkpoint protein that plays a crucial role in regulating T-cell activation. By competing with the stimulatory receptor CD28 for binding to B7 molecules (CD80 and CD86) on antigen-presenting cells, CTLA4 downregulates immune responses, thus maintaining self-tolerance and preventing autoimmunity. CTLA4 is a key target in cancer immunotherapy, particularly for drugs like ipilimumab, which aim to enhance anti-tumor immunity by blocking this checkpoint and activating T cells.

The expression of CTLA4 in this cell line was confirmed using flow cytometry with a target-specific antibody, ensuring reliable and consistent receptor density across the cell population.

37°C, 5% CO₂, humidified atmosphere.

After thawing, split the cells at a ratio of 1:2 to 1:3 in T25 flasks and allow the cells to recover from the freezing process and to adhere (for adherent cultures) for at least 24 hours.

1. Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit.

2. Upon receipt, either store the cryovial immediately at temperatures below -150°C to ensure the preservation of cellular integrity, or proceed to step 3 if immediate culturing is required.

3. For immediate culturing, swiftly thaw the vial by immersing it in a 37°C water bath with clean water and an antimicrobial agent, agitating gently for 40-60 seconds until a small ice clump remains.

4. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening.

5. Carefully open the disinfected vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of room-temperature culture medium, mixing gently.

6. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium.

7. Gently resuspend the cell pellet in 10 ml of fresh culture medium. For adherent cells, divide the suspension between two T25 culture flasks; for suspension cultures, transfer all the medium into one T25 flask to promote effective cell interaction and growth.

8. Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.

Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods.

To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections.

For long-term preservation, place vials in vapor-phase liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable only as a short interim step before transfer to liquid nitrogen.

Cryopreserved cell lines are shipped on dry ice in validated, insulated packaging with sufficient refrigerant to maintain approximately −78 °C throughout transit. On receipt, inspect the container immediately and transfer vials without delay to appropriate storage.

At Cytion, we stand behind the quality and viability of our products. We guarantee that our cells will be viable at the start of culture for 60 days after shipment from our facilities. We assure that all materials conform to the specifications described in the Certificate of Analysis and/or Product Sheet.

For full details, please refer to our Warranty Statement.