Materials and Reagents

1. 10 cm sterile Petri dishes (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875711Z )
   
2. 1.5 ml tubes (Eppendorf, catalog number: 022363204 )
   
3. 35 mm TC-Treated culture dish (Corning, catalog number: 430165 )
   
4. Falcon round-bottom polystyrene 5 ml tubes (Corning, Falcon^®, catalog number: 352003 )
   
5. Sterile 40 μm cell strainer (Corning, catalog number: 431750 )
   
6. 35 mm glass bottom dish (Cellvis, catalog number: D35-20-1-N )
   
7. 3 ml transfer pipette (Sigma-Aldrich, catalog number: Z135003 )
   
8. Zebrafish adults (wild-type or mutant strains)
   
9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
   
10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
    
11. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C5080 )
    
12. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: 63138 )
    
13. ddH₂O (Sigma-Aldrich, catalog number: W4502 )
    
14. DMEM/F-12 medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 11330-032 )
    
15. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 16000-036 )
    
16. Tricaine (Sigma-Aldrich, catalog number: A5040 )
    
17. 10x trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 15400-054 )
    
18. Tris-EDTA buffer solution (pH 9.0) (Sigma-Aldrich, catalog number: SRE0063 )
    
19. Collagenase P (Sigma-Aldrich, catalog number: COLLP-RO )
    
20. Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010-023 )
    
21. Hank's balanced salt solution (HBSS), with calcium and magnesium, no phenol red (HBSS/Ca/Mg) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14025-092 )
    
22. MitoSOX^TM red mitochondrial superoxide indicator (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: M36008 )
    
23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
    
24. 7-aminoactinomycin D (7-AAD) (BD, BD Pharmingen^TM, catalog number: 559925 )
    
25. Annexin V-APC (BD, BD Pharmingen^TM, catalog number: 550474 )
    
26. Annexin V binding buffer, 10x concentrate (BD, BD Pharmingen^TM, catalog number: 556454 )
    
27. CellROX^® green reagents, for oxidative stress detection (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C10444 )
    
28. CellROX^® green flow cytometry assay kit (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C10492 )
    
29. Certified^TM low-melting agarose (Bio-Rad Laboratories, catalog number: 1613111 )
    
30. Instant Ocean aquarium sea salt mixture (Spectrum Brand, Instant Ocean, catalog number: SS15-10 )
    
31. 24x tricaine solution (see Recipes)
    
32. DMEM/F-12 medium (see Recipes)
    
33. 60x E3 embryo medium stock solution (see Recipes)
    
34. Homogenization medium (see Recipes)
    
35. 5 mM MitoSOX stock solution (see Recipes)
    

Equipment

1. Dissecting forceps - microdissection (DR Instruments, catalog number: 18 )
   
2. Ultrafine single deer hair with handle (Ted Pella, catalog number: 119 )
   
3. Thermoblock (Sigma-Aldrich, catalog number: Z605271 )
   
4. Manual pipette PR-200 (Mettler-Toledo, Rainin, catalog number: 17008652 )
   
5. Standard tabletop centrifuges (Eppendorf, catalog number: 022620401 )
   
6. Dissecting microscope (Leica, model: Leica S8 APO )
   
7. FACS-Fluorescence-activated cell sorter [e.g., FACSAria II equipped with FACSDiva software using the blue 488-nm and red 640-nm lasers (BD)]
   
8. Confocal microscope [e.g., LSM 510 NLO Meta system mounted on an Axiovert 200M microscope (Carl Zeiss) with a Plan-Apochromat 10x/0.45 or a Plan-Apochromat 20x/0.75 objective lens]
   

Software

1. FACSDiva software

Procedure