• Grape juice agar plates
Prepare grape juice agar plates as follows:
- Autoclave 25-30 g of agar with 700 ml H2O for 40 min (solution A).
- Add 20 ml of 95% ethanol to a 50-ml tube containing 0.5 g of methyl 4-hydroxybenzoate (Sigma-Aldrich, H3647). Add the solution of methyl 4-hydroxybenzoate to 300 ml of 100% grape juice concentrate (solution B).
- Add solution B to solution A, and pour the mixture into 10-cm plastic dishes.
Tip: Do this step quickly to avoid the solidification of the mixture in the pouring container.
Tip: An extra number of agar dishes should be prepared in case there is a peak of egg-laying.
• Household bleach (sodium hypochlorite solution)
• Embryo washing buffer: 0.7% NaCl, 0.05% Triton X-100, autoclaved.
• Sterilized water: autoclaved MilliQ water
• Schneider's Drosophila medium (Gibco, 21720024)
• Fetal bovine serum for insect cell culture (FBS, Sigma-Aldrich, F0643)
• Cell-Tak (Corning, 354240)
• Sodium bicarbonate (Sigma-Aldrich, S5761)
• Sodium hydroxide (Sigma-Aldrich, S2770)
• Injection compounds (or Seahorse XF Cell Mito Stress Test Kit, Seahorse Bioscience, 103015-100) (See Table 1)
Port A: 50 µM oligomycin (Sigma-Aldrich, 75351; dissolved in 95% ethanol to make a 5 mg/ml (6.32 mM) stock solution)
Port B: 10 µM FCCP (Sigma-Aldrich, C2920; dissolved in 95% ethanol to make a 50-mM stock solution)
Port C: 50 µM antimycin A (Sigma-Aldrich, A8674; dissolved in 95% ethanol to make a 40-mM stock solution) and 50 µM rotenone (Sigma-Aldrich, R8875; dissolved in 95% ethanol to make a 2-mM stock solution)
Note: Compounds should be dissolved in serum-free Schneider's Drosophil medium.
RIPA lysis buffer: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%(w/v) Na deoxycholate, 2 mM EDTA, 1%(w/v) NP-40, 0.1% SDS
• 10-cm plastic dishes (Petri dishes)
• A sufficient number of Drosophila lines (the ratio of females to males should be approximately 2 to 1) to collect over 1,200 eggs per line.
Note: The number of obtained eggs may vary according to fly genotypes. We collected 800 - 1,000 eggs (embryos) from 1,000 fly pairs.
Tip: Approximately 5 x 104 cells can be obtained from 100 embryos.
• 1.5-ml Eppendorf-type tubes (sterilized)
• Embryo-collecting container (tailor-made, 7.5 cm in internal diameter, 11.5 cm in height, See Figure 2a, b)
Note: One container can accommodate approximately 600 flies.
• Dounce tissue homogenizer with a loose pestle (working volume of 1 ml)
• Cotton swabs
• 40-µm cell strainer (Falcon, 352340)
• 50-ml Falcon tubes (Falcon, 352070)
• 15-ml Falcon tubes (Falcon, 352096)
• BCA protein assay kit (ThermoFisher, 23225)
• Seahorse XF24 FluxPak Mini (Agilent 100867-100, Figure 3). Contents include
6 XF24 sensor cartridges (each set contains a sensor cartridge, hydro-booster, 24-well utility plate and lid), 10 XF24 cell culture microplates, and 1 bottle (500 ml) of Seahorse XF Calibrant Solution.