Tunel kit

Apoptotic cells were detected in situ using the Roche TUNEL kit (Roche, Mannheim, Germany) according to the manufacturer's protocol. The TUNEL assay was conducted to visualize the 3'-OH ends of DNA fragments in apoptotic cells. After xylene dewaxing, sections were rinsed three times in distilled water for 5 min, and they were washed in methanol containing 0.3% H₂O₂ at room temperature for 30 min to inhibit endogenous peroxidase activity. After rinsing in PBS three times at room temperature for 5 min, sections were treated with proteinase K at 37 °C for 6 min. Section were rinsed in PBS three times at room temperature for 3 min, were soaked in TdT buffer for 10 min, and incubated in 50 μl TdT buffer containing TdT at 37 °C for 60 min in a moist chamber (Roche). After three rinsing steps in PBS at room temperature for 5 min, the sections were incubated in 50 μl FITC (Roche) at 37 °C for 40 min. After three further rinses in PBS for 3 min, the brain sections were incubated in DAB (Roche) at room temperature for 3 min, and the signal was observed using a confocal microscope (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany).^{129 (link)}

TUNEL labeling was performed in tissue sections using the Roche TUNEL kit (Roche, 12156792910) according to the manufacturer’s protocol. A mounting medium with DAPI (UltraCruzTM, Santa Cruz Biotechnology, Dallas, USA, sc-24941) was placed over the tissue, followed by a coverslip. Tile images were taken with an epifluorescent microscope (Zeiss Axio Imager M2, Zeiss, Germany). DAPI signal (blue) was overlayed with Texas Red (TUNEL⁺ cells) and quantified using ImageJ to assess the number of TUNEL⁺ cells overlapping with DAPI in the areas of interest.

Cell death was assessed in tissue sections using terminal deoxynucleotidyl transferase-mediated dUTP nick- end labeling (TUNEL, Roche TUNEL kit (12 156 792 910; Roche, Basel, Switzerland)), as previously described (Cade et al., 2014 (link); Paschalis et al., 2017 (link)). Mounting medium with DAPI (Ultra-Cruz; sc-24 941; Santa Cruz Biotechnology) was placed over the tissue, followed by a coverslip. Tile images were taken using an epifluorescent microscope (Zeiss Axio Imager M2; Zeiss). DAPI signal (blue) was overlayed with Texas red (TUNEL+ cells) and quantified with ImageJ software version 1.43 or above (NIH; http://imagej.nih.gov/ij) to assess the number of TUNEL+ cells overlapping with DAPI in the areas of interest. At least three different tissue sections per eye were analysed, and data were presented as a percentage of the total DAPI area. Quantification of TUNEL+ cells was preformed centrally at 1/3rd of the diameter of the retina and peripherally at 2/3rds of the diameter.

The Dead End Fluorometric TUNEL system (Roche TUNEL kit; Roche Applied Science, Burgess Hill, UK) was used as described by the manufacturer. Briefly, paraffin sections were prepared as described by the manufacturer and subjected to TUNEL analysis after incubation with proteinase K; Hoechst counterstaining was then performed. Fluorescence images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan), and images were obtained using Zeiss software version 4.6. Apoptotic cells within the retina fluoresced red.

At the end of MLR performed in the presence of EV from dnIKK2-Treg or Tact or Trest, cell cycle was evaluated by FACS analysis of propidium iodide (PI) incorporation. Briefly, T cells were fixed with 70% ethanol overnight, washed with PBS, incubated with PI (10 μg/mL) and RNase A (100 μg/mL) and FACS-analyzed. Cell division number was measured by FACS evaluation of CFSE labeled cells on vital (7-AAD⁻) cells, followed by analysis with FlowJo software. Apoptosis was evaluated by FACS using the TUNEL method (TUNEL Kit, Boehringer Mannheim, Mannheim, Germany), as previously described^{62 (link)}. In selected experiments apoptosis was evaluated by AnnexinV/7AAD staining (PE AnnexinV Detection Kit, BD Pharmingen) and FACS analysis.

Cel was purchased from Shanghai Yuanye Company (purity >97%); high‐fat feed was provided by Chengdu Dossy Experimental Animal Co. Ltd.; detection kits for total cholesterol (TC), triacylglycerol (TG), high‐density lipoprotein cholesterol ( HDL‐C) and low‐density lipoprotein cholesterol (LDL‐C) were provided by Shanghai Mind Biological Engineering Co., Ltd.; detection kits for aspartate aminotransferase (GOT), alanine aminotransferase (GPT), superoxide dismutase (SOD), malondialdehyde (MDA) were provided by Nanjing Jiancheng Bioengineering Research Institute; terminal in situ nick end labeling (TUNEL) kit was provided by Boehringer Mannheim; and anti‐Nrf‐2, OH‐1 and GAPDH antibodies were purchased from Abcam.