Sytox deep red

IF staining was carried out as previously described [58 (link)]. For IHF, cryosections were prepared from colon tissues frozen in O.C.T Compound (VWR), fixed in 4% paraformaldehyde (PFA; MilliporeSigma) at 4 °C for 8 min, and blocked in 2% bovine serum albumin (BSA; VWR) at room temperature for 30 min. The blocked sections were incubated with primary antibodies at 4 °C overnight, followed by 3-time rinses in PBS and subsequent incubation with secondary antibody at room temperature for 30 min. The slides were washed in PBS 3 times, air dried, and mounted with Prolong gold antifade mountant with SYTOX deep red (ThermoFisher). Anti-occludin rabbit antibody (ProteinTech, 27,260–1-AP; 1:400), anti-claudin1 rabbit antibody (ProteinTech, 13,050-1-AP; 1:1000), and goat anti-rabbit-Alexa Fluor-488 secondary antibody (Invitrogen, A-11008; 1:200) were used for both IF and IHF. Quantitative analysis of IF and IHF images was conducted using ImageJ software.

A cell viability assay was carried out by direct counting of cells (live/dead) based on the laser confocal observations performed in FluoroBrite™ DMEM (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) media. Calculations were made by direct observations, and images were captured and processed using NIS Elements software (Nikon, Tokyo, Japan). Live cells were visualized by fluorescein diacetate (FDA, Thermo Fischer Scientific, Waltham, MA, USA), and dead cells were visualized by nuclei labeling using Sytox Deep Red (Thermo Fischer Scientific, Waltham, MA, USA).
For the MTT test, cells were placed in a 96-well plate (~1500 cells per well) and incubated under standard conditions for 3 days. Lipofuscin loading and irradiation were performed as described in Section 2.2.2 and Section 2.2.3. At 1 and 48 h after the irradiation, 20 μL of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution (5 mg/mL in PBS) was added, and the samples were incubated at 37 °C for 4 h. The formed formazan crystals were dissolved in DMSO and assessed colorimetrically at 550 nm using an Infinite 200 Pro plate reader (Tecan, Zürich, Switzerland).
The statistical analysis of the data on cell viability was performed by two-way ANOVA with Tukey’s multiple comparison t-test using Prism software (GraphPad, Boston, MA, USA).