Materials and Reagents

1. (Optional) Keep-warm bag
2. Sterile sampling tubes (the volume size should be within 0.2 from 1.5 ml) (e.g., 0.2 ml tube, Bio-Bik, catalog number: 133003 ; 1.5 ml tube, VIOLAMO, catalog number: 1-1600-1 )
3. 35 mm dishes (e.g., IWAKI, catalog number: 1000-035 )
4. DMEM without Phenol Red (Sigma-Aldrich, catalog number: D2902 )
5. DMEM (Nacalai, catalog number: 08456-94 )
   Although DMEM from other suppliers is usable as a substitute for these DMEMs, the possibility cannot be excluded that the difference in concentrations of minor ingredients affects success rates and results slightly.
6. Sodium bicarbonate (Sigma-Aldrich, catalog number: S8761 )
7. HEPES (Nacalai, catalog number: 17557-94 )
8. D-Glucose (Sigma-Aldrich, catalog number: G8769 )
9. Penicillin/streptomycin (Thermo Fisher Scientific, catalog number: 15070-063 )
10. L-Glutamin (Nacalai, catalog number: 16948-04 )
11. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
12. Luciferin (WAKO, catalog number: 126-05116 )
13. Silicone (Shin-Etsu, catalog number: KS-64 )
    Note: Reagents #6-13: Similar items from other suppliers serve as a substitute.
14. Adenovirus vectors carrying the luciferase (luc) gene driven by circadian promoter/enhancer elements (e.g., Bmal1-luc, Per2-luc and Per3-luc). Some entry vectors we constructed and deposited are available from the RIKEN bank as follows:
    RDB15083 mouse Period2 (-2.8 ~ +0.1 kb) - Luciferase/pENTR-1A
    RDB15084 human Period3 (-3.1 ~ +0.2 kb) - Luciferase/pENTR-1A
    RDB15085 human Bmal1 (-1.7 ~ +0.1 kb) - Luciferase/pENTR-1A
    The ViraPower Adenoviral Gateway Expression Kit (K4940-00, Thermo Fisher Scientific) is required to generate adenoviruses using these entry vectors. Procedures using adenoviruses must be performed inside P2 areas. Autoclave and discard any infectious wastes
15. Infection medium (see Recipes)
16. Pre-incubation medium (see Recipes)
17. Rinse medium (see Recipes)
18. Luciferin-containing medium (see Recipes)
    

Equipment

1. Non-slip, cosmetic-use tweezers (e.g., Shiseido, eyebrow nippers 211)
2. Tweezers (e.g., FST, catalog number: 18132-12 )
3. Block incubator set to 36.5 °C (e.g., ASTEC, model: BI-516C )
   
4. Laminar flow cabinet (e.g., SANYO, model: MCV-B131F )
   Note: Equipment #1-4: Similar items from other suppliers serve as a substitute.
5. Incubator at 35-36.5 °C with 5% CO₂ (e.g., ASTEC, model: SCA-165DRS )
   Use a CO₂ incubator that utilizes an infrared sensor that is not affected by humidity inside the chamber (see details in Procedure Step 18).
6. Photomultiplier tube (Hamamatsu, model: LM2400 )
7. Luminescence microscope (Olympus, model: LV200 )
   

Software

1. Cosinor software (freely downloadable from a website: https://www.circadian.org/softwar.html)

Procedure