Materials and reagents

Biological materials

1. Drosophila melanogaster (collected from Bloomington Drosophila stock center)

Reagents

1. DCF ROS/RNS assay kit (Abcam, catalog number: ab238535); contains enough reagents to perform 96 assays (Table 1)

Table 1. Storage conditions, concentrations, and volume of individual components from the kit

2. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-50G)

3. Bradford reagent (Thermo Scientific, catalog number: 23238)

4. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)

5. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333)

6. Disodium phosphate (Na₂HPO₄) (Apex, catalog number: 20-147)

7. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655)

Solutions

1. 1× stabilization solution (see Recipes)

2. 1× catalyst (see Recipes)

3. 1× DPS (see Recipes)

4. Hydrogen peroxide standard (see Recipes)

5. BSA standard (see Recipes)

6. 1× phosphate buffer saline (see Recipes)

Recipes

Note: Recipes below are for 50 reactions (one reaction per well of a 96-well plate). Prepare a little extra volume than the exact volume required to make up for any pipetting error.

1. 1× stabilization solution (5.5 mL)

Mix 550 μL of 10× stabilization solution (from the assay kit) with 4,950 μL of deionized water to make 1× solution. Vortex and store at 4 °C until use.

2. 1× catalyst (2.75 mL)

Mix 11 μL of 250× catalyst (from the assay kit) with 2,739 μL of 1× PBS (Recipe 6) to prepare 1× catalyst solution. Vortex and use immediately.

3. 1× DPS (2.75 mL)

Mix 27.5 μL of DCF-DiOxyQ with 110 μL of priming reagent. Vortex and incubate at room temperature for 30 min. Then, add this mixture to 5362.5 μL of 1× stabilization solution (Recipe 1). Cover the tube in aluminum foil. Store on ice for immediate use. Any excess can be stored at -20 °C for up to a week for future assays.

4. Hydrogen peroxide standards

Critical: Freshly prepared sets of standards are made in tubes wrapped with aluminum foil. Prepare a 2 mM H₂O₂ stock solution by mixing 1 μL of 4,400× H₂O₂ with 4.4 mL of deionized water. This stock solution is used to prepare standards. See Table 2 for H₂O₂ standard preparations with 1× PBS (Recipe 6).

Table 2. Hydrogen peroxide standards

Note: In this protocol, the standard curve for hydrogen peroxide has been adjusted from the original range of 0.039 μM to 0.313 μM, as specified in the protocol for the ab238535 kit (Abcam). This modification is based on the observation that the lower concentration range (0.039 μM) is not necessary for the sensitivity and detection limits of the assay under the conditions used in this study. In our sample, the free radical concentration equivalent to H₂O₂ concentration in the standard curve fell between 10 and 20 μM. However, researchers can use lower concentrations of H₂O₂ in the standard curve as suggested by the Abcam protocol if they use a lower number of flies or a higher dilution of fly samples.

5. BSA standards

Prepare 2 mg/mL BSA stock solution by combining 40 μL of BSA stock (50 mg/mL) with 960 μL of 1× PBS (Recipe 6), yielding a total volume of 1,000 μL. Then, prepare a series of standard dilutions from the 2 mg/mL BSA stock solution with varying concentrations in cooled 1× PBS as shown in Table 3.

Table 3. BSA standards

6. 1× phosphate buffer saline (PBS)

a. Begin by making a 10× PBS solution, placing 250 mL of distilled water in a 500 mL container.

b. Add the following reagents to the distilled water:

• 80.06 g of NaCl (1.37 M)

• 2.01 g of KCl (27 mM)

• 26.8 g of Na₂HPO₄ (100 mM)

• 2.45 g of KH₂PO₄ (18 mM)

c. Adjust the pH of the solution to 7.4 using a pH meter.

d. Add distilled water to bring the total volume up to 500 mL.

e. Aliquot prepared 10× PBS into five bottles, each containing approximately 100 mL of PBS. Sterilize by autoclaving for 30 min at 121 °C.

f. Dilute the 10× PBS stock solution 1:10 with distilled water. For example, mix 50 mL of 10× PBS with 450 mL of distilled water to make 500 mL of 1× PBS.

Laboratory supplies

Note: Similar lab supplies from other companies will work as well.

1. 96-well transparent plate (Genesee Scientific, catalog number: 25-109)

2. 96-well black plate (Thermo Scientific, catalog number: 165305)

3. PolyPestle (Genesee Scientific, catalog number: 25-280)

4. Microcentrifuge tube (clear polypropylene 1.7 mL tube) (Olympus, catalog number: 24-282)

5. Single (Sartorius brand, 1–10, 10–100, 20–200, 100–1,000 μL) pipette and multi-channel pipettes (Poseidon 8 Channel P300 Pipettor, 20–300 μL)

6. Sterile tips (Olympus brand, 10, 100, 200, and 1,000 μL tips)

7. Pasteur pipette (VWR, catalog number: 14672-200)

Equipment

1. Agilent BioTek Synergy H1; filter set: excitation at 480 nm and emission at 530 nm

Note: Any other plate reader that can measure the above fluorescence wavelengths can be used.

2. Microcentrifuge (Hermle, catalog number: Z216-MK)

3. Vortex (Benchmark, catalog number: BV1000)

4. Mini centrifuge (Genesee Scientific, catalog number: 27-523)

5. Autoclave (Priorclave, catalog number: 150-320L)

6. -80 °C freezer (Eppendorf, catalog number: EPP: F740540015)

7. -20 °C freezer (VWR, catalog number: 76580-128)

8. pH meter (Thermo Scientific, model: Orion Star A211)

Software and datasets

1. GraphPad Prism (https://graphpad-prism.software.informer.com/7.0/)

2. Gen5 3.14 ( https://www.agilent.com/en/support/biotek-software-releases)

3. Microsoft Excel

Procedure