Daf 2 da

As an indicator of NO presence, DAF-2 DA was used similarly as in our previous study [30 (link)]. Whole blood was diluted 1:9 (v:v) in modified physiological salt solution (in mmol/L: NaCl 118.99, KCl 4.69, NaHCO₃ 25, MgSO₄.7H₂O 1.17, KH₂PO₄ 1.18, CaCl₂.2H₂O 2.5, Na₂EDTA 0.03, glucose 5.5, pH 7.4, all reagents provided by Sigma-Aldrich, St. Louis, MO, USA) and treated with DAF-2 DA (25 μmol/L, Abcam, Cambridge, UK) at room temperature for 10 min in the dark. Blood samples were analyzed under a fluorescence microscope (Axio Imager M2, Zeiss, Jena, Germany) using filters for fluorescein isothiocyanate (λ_ex = 465–495 nm, λ_em = 515–555 nm). The fluorescence was quantified using ImageJ 1.53e software (National Institutes of Health, Bethesda, MD, USA). More precisely, RBCs were separated using a threshold adjuster in computer-recorded images. Afterwards, all of them were filtered according to their size and circularity. RBCs passing or touching through the image edges were excluded automatically. Overlapping RBCs were removed from the analysis manually. We determined fluorescence of an average of 860 RBCs in each sample. The intensity of fluorescence is presented as integrated density corresponded to a single RBC.

J774 were stained for the detection of ROS production using 2′,7′-Dichlorofluorescin diacetate (DCFDA; 15 µM; Sigma-Aldrich), for intracellular nitric oxide (NO) using diaminofluorescein-2 diacetate (DAF-2 DA; 2,5 µM; Abcam, Cambridge, UK) or for mitochondrial membrane potential using MitoTracker™ Red CMXRos (100 nM; Thermo Fisher) according to the manufacturer’s instructions for 30 min at 37 °C. Cells were harvested and washed in PBS/0.5% BSA. A total of 50 000 cells were analyzed after the exclusion of dead cells and debris. Five minutes before measurement, Hoechst 33258 fluorescent dye was added and used to exclude dead cells. Data were collected using the LSR II cytometer and analyzed using GateLogic 400.2 A software. Representative dot plots and histograms illustrating the gating strategy are shown in Supplementary Figure S2.

Isolated lower limb muscle was minced and digested with collagenase type IV (Worthington; final concentration, 2 mg/mL) and 2 mM CaCl₂ in PBS at 37°C for 45 minutes. Cells were fixed using 0.1% PFA for 10 minutes at room temperature (RT), followed by permeabilization using 0.1% Triton X-100 (Thermo Fisher Scientific) for 5 minutes at RT. Unspecific binding was blocked using FcR receptor blocking reagent (Miltenyi Biotec; 130-092-575), followed by incubation with allophycocyanin-labeled primary antibodies against CD31 (CD31-APC; clone MEC13.3; BioLegend; 102510; 1:100). To visualize NO, 1 × 10⁶ unfixed cells were incubated with fluorescent NO probe 1.5 μM DAF-2 DA (Abcam; ab146631) at 37°C for 35 minutes, based on preliminary dose-finding studies and as published previously (69 (link)). Cells were washed and immediately analyzed using flow cytometry (BD Biosciences FACSCanto II). Forward and side scatter gates were set to exclude debris and cellular aggregates. Unstained cells were used as a negative controls.