Quantifying Intracellular ROS in Myotubes

To determine intracellular ROS production, differentiated C2C12 myotubes were incubated with the fluorogenic dye 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) (D6883; Sigma‐Aldrich), which is oxidized by ROS to produce DCF and emits green fluorescence from the dye (Kim & Xue, 2020 (link)). Cells were plated at a density of 1.3 × 10⁵ cells per well in a 24‐well plate and randomly divided into the following groups: the Veh, AII, AII + 100 nM VD₃, AII + 10 nM 1,25VD₃, and AII + Lo groups. All treatments were applied at the differentiation phase as follows: 1 μM AII at Days 4–6, 100 nM VD₃ and 10 nM 1,25VD₃ at Days 5–6 and 10 μM losartan applied 2 h before AII was administered on Day 4. Myotubes incubated overnight with 20 μM hydrogen peroxide (H₂O₂) (88597; Millipore, Temecula, CA, USA) were used as a positive control. On Day 7, DCF‐DA staining was performed according to a previous report (Kim & Xue, 2020 (link)) with slight modifications and all procedures were protected from light. After the culture medium was removed and washed once with warm phenol red‐free DMEM (17‐205‐CV; Corning), the myotubes were incubated with 10 μM DCF‐DA solubilized with methanol in phenol red‐free DMEM at 37°C in a 5% CO₂ incubator for 30 min in the dark. The medium containing DCF‐DA was then removed, and the cells were washed once with phenol red‐free DMEM and twice with PBS. Representative fluorescence images using the FITC channel were captured with an Olympus Inverted Fluorescence Microscope Model IX83 (Olympus, Tokyo, Japan) equipped with an ORCA‐Flash 2.8 Digital CMOS Camera (C11440) (Hamamatsu Photonics, Hamamatsu, Japan) at ×100 magnification.
To measure fluorescence intensity using a microplate reader, protein was extracted from treated myotubes using ice‐cold RIPA buffer containing a cocktail of protease inhibitors (P8340; Sigma‐Aldrich). The extracted proteins were centrifuged at 21,130 g for 10 min (4°C) and 100 μL of the supernatant was transferred to a black clear bottom 96‐well plate (3603; Corning). The fluorescence intensity was measured using a multimode microplate reader (Infinite® M200 PRO; Tecan Trading AG, Männedorf, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A BCA assay (Thermo Scientific) was performed to determine the protein concentration, which was used to normalize the DCF fluorescence intensity.