Accuri c6 flow cytometry system

SAβG activity was measured using Senescence Cells Histochemical Staining Kit (Sigma-Aldrich), according to the manufacturer’s recommendations and by flow cytometry using C₁₂FDG^{44 (link)}. For the SAβG staining, cells were fixed with 1× fixation buffer for 7 min at RT, washed 2× with phosphate-buffered saline (PBS) and incubated with the staining mixture (containing X-gal) at 37 °C (without CO₂) for 5 h. Images were taken using a Nikon TMS-F microscope equipped with GXCAM LITE live camera and the percentage of SAβG-positive cells was quantified using ImageJ software. For flow cytometry, control and SIPS cells were pre-treated with either 100 nM of pH modulator-Bafilomycin A1 for 1 h followed by incubation with 33 µM of C₁₂FDG for an additional 1 h before analysis on BD Accuri C6 flow cytometry system (BD biosciences). For the staining of the ligated carotid arteries, OCT-embedded pre-fixed tissue sections were washed 2× with PBS and incubated with the SAβG staining mixture in a humidified chamber overnight at 37 °C. The next day, sections were washed 2× with PBS, and images were taken using an Olympus BX51 microscope equipped with an Olympus Lumenera Infinity 3 digital camera. The same section was then imaged by confocal microscopy to identify VSMC-lineage traced cells in the SAβG-positive areas of the tissue.

After clone selection cells were expanded, cells were dissociated, washed once with DPBS and then spun for 5 min at 200 g. After spin, supernatant was discarded, and cells were resuspended for staining in 100 μl DPBS buffer containing 2 % FBS. Cells were then divided into two separate Eppendorf tubes with 100 μl of cells in each one. Afterwards, staining was done with fluorophore-conjugated antibodies (Table 2) for 20 min at 4 °C. Cells were resuspended in 250 μl PBS wash and then analyzed with a BD Accuri C6 Flowcytometry system (BD Biosciences).

The population of putative spermatogonial stem cells (SSCs) in the cells in culture was estimated as the MHC I/CD9+/CD49f+ population [54 (link),55 (link),56 (link),57 (link),58 (link),59 (link)] using a BD Accuri C6 Flow cytometry system without sorting. BD antibodies were used (Supplementary Table S2) at a concentration of 5 µL of antibody per 50,000 cells in 100 µL. Cells were incubated with the antibody for one hour at room temperature and then washed with FACS Buffer (1% FBS in PBS). The obtained data were analyzed using BD Accuri software. Unstained cells and isotype controls (Supplementary Table S2) were used to optimize channel compensation and as a negative control. In every condition, at least 10,000 events were evaluated.
In separate experiments, the same staining method was used to perform Fluorescence-Activated Cell Sorting (FACS) using BD FACS ARIA. Cells expressing MHC I-/CD9+/CD49f+ were sorted and used for DNA FISH analysis.

The iPSCs were harvested using TrypLE Express enzyme (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde for 10 mins at room temperature and then washed with DPBS. Before fluorescence-activated cell sorting analysis, cells were permeabilized with 0.2% Tween-20 in DPBS for 10 mins at room temperature and stained with fluorophore-conjugated antibodies for 1 h at 4 °C on a shaker. Relative fluorophore-conjugated animal nonimmune Immunoglobulin were used as the negative control (Antibodies and nonimmune immunoglobulin used are listed in Table 2). Cells were then analyzed on a BD Accuri™ C6 Flow Cytometry system (BD Biosciences).

Oxidative stress was assessed by flow cytometry using two markers, reactive oxygen species (ROS) and intracellular glutathione (GSH) (ImmunoChemistry Technologies, Burlington, ON, Canada). Firstly, cells were cultivated for 24 h with or without anethole. Second, total ROS Green was reconstituted with DMSO, diluted in the buffer, added to each sample (10⁶ cells/mL), and incubated in the dark for 1 h at 37 °C. The key reagent can then be detected by flow cytometry depending on presence of ROS. Lastly, a decrease of intracellular GSH, a molecule with reducing power playing a key role against free radicals and toxins, is an indicator of the induction of apoptosis in cells stressed by oxidation. To evaluate levels of intracellular GSH, ThioBright™ Green reagent was reconstituted in DMSO, added to the cells suspension (1:200), and incubated in the dark for 30 min. After washing with PBS twice, the intensity of fluorescence was analyzed by BD Accuri C6 Flow Cytometry system (BD Bioscience) and the percentage of positive cells was calculated. Each experiment was repeated three times.