Materials and Reagents

1. 24-well cell culture plate (Corning, Costar^®, catalog number:3524)

2. Cell culture flask, T25 (Corning, Costar^®, catalog number:430639)

3. Round coverslip, 9 mm (Biosharp, catalog number: BS-09-RC), place the coverslips in a 75% ethanol-containing centrifuge cube

4. Adhesion microscope slides (CITOTEST, catalog number: 80312-3161)

5. Parafilm (Bemis, catalog number:4157)

6. For cell preparation:

   1. Dulbecco’s Modified Eagle Medium (DMEM), 1g/L D-Glucose, with L-Glutamine (Gibco, catalog number: C11885500BT). Store at 2-8°C for 1 year

   2. Fetal Bovine Serum (FBS) (Gibco, catalog number:10099141). Aliquot and store at -20°C

   3. Penicillin-Streptomycin (10,000U/ml) (Gibco, catalog number:15140122). Store at -20~-5°C for 1 year

   4. Phosphate Buffered Saline (PBS), sterile-filtered (Servicebio, catalog number: G4202). Store at 2-30°C for 18 months

   5. 0.25% Trypsin-EDTA, (Gibco, catalog number: 25200-072). Store at -20~-5°C for 24 months

7. Phosphate buffered saline (PBS) powder (Absin, catalog number: abs9266). Store at room temperature (RT) for 2 years

8. 4% paraformaldehyde (PFA) (Solarbio, catalog number: P1110). Aliquot and store at -20°C for 1 year, and avoid repeated freezing and thawing. Store at 4°C for up to a month

9. Triton X-100 (Solarbio, catalog number: T8022). Store at RT for 2 years

10. Normal goat serum (Solarbio, SL038). Store at -20°C for 5 years

11. Primary antibodies:

    1. MRPL12 antibody (Novas, catalog number: 3B12-1A3). Aliquot and store at -20°C. Avoid freeze-thaw cycles

    2. Nrf2 antibody (Abcam, catalog number: ab62352). Aliquot and store at -20°C. Avoid freeze-thaw cycles

12. Secondary antibodies:

    1. Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A32723). Store at 4°C in the dark

    2. Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Thermo Fisher Scientific, Invitrogen, catalog number: A32754). Store at 4°C in the dark

13. Hoechst 33342 (Life technologies, catalog number: HH-H3570). Aliquot and store at -20°C in the dark

14. Mounting medium, antifading (Solarbio, catalog number: S2100). Store at 4°C for 1 year

15. Cell culture medium (see Recipes)

16. PBS (see Recipes)

17. Permeabilization solution (see Recipes)

18. Blocking solution (see Recipes)

Equipment

1. Tweezers

   Note: The tweezers should be sterile when conducting cell preparations. The subsequent steps do not require sterile conditions.

2. Paper towel

3. (Optional) Wet chamber

4. (Optional) Refrigerator (Haier, model: BCD-328WDPT)

5. Constant temperature incubator (Panasonic, model: MIR-262-PC)

6. Nikon inverted fluorescence microscope (Nikon, model: Ti-S)

7. Clean bench (Airtech, model: SW-CJ-1FD)

8. Directly heated CO₂ incubator (Thermo Fisher Scientific, model: 240i)

Software

1. ImageJ

2. Prism GraphPad

Procedure