Tunel assay kit

TUNEL assay kit (Sigma Aldrich, 12156792910) was used to label double stranded DNA breaks. Sections were co-labeled with DAPI and with antibody to lamin B or pH2AX.

RPE cell death was detected using a TUNEL assay kit (Sigma-Aldrich, UK) according to the manufacturer's instructions. In brief, RPE cells plated for experiments with oxPOS or 4-HNE were washed with cold PBS and then fixed with 4% paraformaldehyde (PFA) for 1 h at 15–25°C before washing with PBS. The samples were then permeabilized with 0.1% Triton-X for 2 min on ice (2–8°C). As a negative control, one well contained labelling solution only. As a positive control, 3 U/ml recombinant DNASE1 was added to one group of cells to induce DNA strand breaks prior to labelling. The complete TUNEL reaction mixture was added to all experimental wells and incubated at 37°C for 1 h. The cells were washed with PBS before imaging under a confocal microscope. A series of 4-HNE concentrations were tested to choose a concentration that induced 50% or less cell death in 48 h treatment (data not shown). Therefore, apart from oxPOS, 5 μM 4-HNE for 24 h was used as an alternative oxidative insult to induce RPE cell death.

Apoptosis was detected using a Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL) assay kit (Sigma-Aldrich, #11684817 910 or #12156792910, St. Louis, MO, United States) according to the manufacturer’s protocols. For the in-vitro experiments, neurons were fixed in 4% paraformaldehyde for 30 min and rinsed with PBS. For the in-vivo experiments, tissue sections were rinsed three times with PBS. After incubation with 3% H₂O₂ for 10 min at room temperature, the neurons and tissue sections were rinsed with PBS, then penetrated with PBS containing 0.2% Triton X-100 and 0.1% sodium citrate for 2 min on ice. For TUNEL staining, the samples were incubated with TUNEL reaction mixture for 60 min at 37°C. After the samples were rinsed three times with PBS, DAPI was used to localize nuclei. A fluorescent microscope was used to obtain images with the detection wavelength at 488 nm or 580 nm. DAPI- and TUNEL-positive cells were counted using ImageJ (n = 5). TUNEL-positive cell ratio = TUNEL-positive cells/DAPI-positive cells.

Apoptosis in cardiac tissue was evaluated using the TUNEL assay kit (Sigma-Aldrich, USA). To determine the ratio of apoptotic nuclei, the total count of TUNEL-positive nuclei was divided by the total count of DAPI-positive nuclei. Each paraffin segment had six random fields evaluated.

Apoptosis was analyzed in the post-cataract surgery explant cultures using a TUNEL assay kit (Sigma Aldrich, St. Louis, MO, USA) that detects double strand DNA breaks, which were co-stained with DAPI and examined by confocal microscopy.

Biocurcumin (BCM-95) and placebo were purchased from DolCas Biotech, LLC (9 Lenel Rd, Landing, NJ 07850, USA). BCM-95 is a capsule containing Biocurcumin-piperine ( Table 1). The placebo is a capsule containing dibasic calcium phosphate (anhydrous). The external beam radiation therapy equipment comprising a linear accelerator was obtained from UNIQUE systems (Varian Medical Systems, California, USA). The brachytherapy equipment used was Microselection HDR from Nucletron (Munchen, Germany). An anti-nuclear factor-kappa B (NF-κB) p65 antibody was purchased from Abcam (1 Kendall Square, Cambridge, USA). A TUNEL assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). A DAPI staining assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA).