Keap1 Knockout cell line （LLC）

Handling procedures

Cell Thawing

• Pre-warm complete culture medium in a 37°C water bath.
• Thaw the cryovial in a 37°C water bath for 1–2 minutes.
• Transfer the cryovial to a biosafety cabinet (BSC) and wipe the surface with 70% ethanol.
• Loosen the cap and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete medium.
• Centrifuge at 125 × g for 5–7 min at room temperature (RT), then discard the supernatant.
• Resuspend the cell pellet in 5 mL of complete medium and transfer the suspension into a T25 flask.
• Incubate the cells at 37°C in a 5% CO₂ incubator.
• Recommended subculturing ratio: 1:2 to 1:3, reaching confluency in 2–3 days.

Cell Passaging

• When cell confluence reaches 80–90%, proceed with passaging.
• Pre-warm complete medium, PBS, and trypsin (0.25% Trypsin-EDTA, Gibco 25200-056) in a 37°C water bath. Once near 37°C, spray the bottles with 75% ethanol and place them in the BSC.
• Retrieve the culture flask from the incubator, spray with 75% ethanol, and transfer it to the BSC.
• To avoid dislodging cells, gently rinse the monolayer with PBS along the upper wall of the flask. Discard the PBS after washing (use 2 mL for T25 flasks).
• Add an appropriate volume of trypsin (1.5 mL for T75, 0.5 mL for T25) and gently tilt the flask to ensure full coverage. Adjust the volume as needed. After 1–2 min, when most cells detach, neutralize digestion by adding an equal volume of complete medium. Gently pipette with a 5 mL serological pipette to ensure complete detachment.
• Transfer the cell suspension to a 15 mL centrifuge tube and spin at 300 × g for 5 min. Discard the supernatant.
• Resuspend the pellet in 5 mL of complete medium, adjust the seeding ratio as required, and replenish the flask with fresh medium (13–15 mL for T75, 5 mL for T25). Add 1% penicillin-streptomycin (dual antibiotic).
• Tighten the cap, gently swirl to mix, and place the flask in a 37°C, 5% CO₂ incubator.

Cell Cryopreservation

• Prepare cryopreservation medium in advance and pre-chill.
• Ensure cells meet freezing criteria: healthy morphology, late-log phase growth, and absence of contamination or senescence (verify under a microscope).
• Digest and centrifuge the cells (refer to the passaging protocol).
• Resuspend the cell pellet in cryopreservation medium (1 mL per vial), mix thoroughly, and aliquot into cryovials.
• Place the vials in a freezing container and store at −80°C overnight.
• For long-term storage, transfer the cryovials to a liquid nitrogen tank.