Nucred live 647

Wing discs were dissected from third instar larvae in Schneider’s insect medium (Sigma-Aldrich), and a small fragment was removed with tungsten needles. To visualize Mmp1 staining, the discs were cultured in Schneider’s insect medium supplemented with 2% heat-activated fetal calf serum, 2.5% fly extract and 5 μg/ml insulin, for 5 hours at 25°C. For P-p38, discs were injured in Schneider’s insect medium and immediately fixed and stained. Ex vivo images were taken using a Leica SPE confocal microscope and processed with Fiji software.
Ex vivo discs were also monitored live after a cut, using puc-Gal4 UAS-GFP as a JNK reporter, using the same medium. For nuclei visualization in ex vivo culture, we used NucRed Live647 (Life Technologies; 1 drop per slide) added 20 minutes before imaging.

Cytotoxicity of Lawsone was measured by phosphorylation at Ser139 residues of the H2A.X histone. Phosphorylation of the H2A.X histone occurs at the site of DNA damage after exposure to polyaromatic hydrocarbons, hydroxyl radicals or ionizing radiation⁶⁹ . 4 h after Lawsone exposure, HEK cells were fixed with 2% paraformaldeyde for 20 min at room temperature (RT) and permeabilized with 0.1% Triton for 5 min at RT. After 30 minutes in blocking buffer, cells were stained with α-phospho-Histone H2A.X (Millipore) for 1 h at RT, followed by staining with the α-rabbit IgG AlexaFluor488 (Dianova,) for 1 h at RT. Nuclei were stained using Nuc red Live 647 (Life technologies). Cell image acquisition and analysis was perfomed using Arrayscan XTI Live High Content Platform (ThermoFisher Scientific).
Formalin-fixed paraffin-embedded skin equivalents were stained either with hematoxylin and eosin (Sigma-Aldrich) or anti-human cornifelin (Sigma) and loricrin (Abcam), followed by anti-rabbit AlexaFluor555 and 488 respectively. Nuclei were counterstained with DAPI. Images were acquired using a Leica DMRB fluorescent microscope and analyzed with ImageJ (https://imagej.nih.gov/ij/).

On the day before imaging, 2,500 cells were plated in each well of an Ibidi 8-well plate chamber (µ-Slide 8 Well high Glass Bottom, Ibidi). After 24 h, cells were washed twice with PBS and stained with NucSpot Live 488 (Biotium) or with NucRed Live 647 (Invitrogen) dyes, following the manufacturer’s instructions. When using tNucSpot Live 488, the dye was incubated with the cells in the imaging solution (Live Cell Imaging Solution (Invitrogen) supplemented with 10% FBS). In the case of NucRed Live 647, cells were washed 4 times with Live Cell Imaging Solution and then imaged in Live Cell Imaging Solution supplemented with 10% FBS. Cells were imaged on an Axio Observer Zeiss wide-field microscope equipped with a Plan-Apochromat 20x/0.8 M27 air objective, incubation chamber with controlled temperature, CO₂ and humidity, and a Hamamatsu camera. Images were acquired every 10 min in the 488 and 647 channels, with the light source X-Cyte lamp at 20% power for 48 h.

2 × 10⁵ A549 cells were seeded on 12-well plates and grown to confluence. A549 cells were stained using the cell-permeant nuclear stain NucRed™ Live 647 (Thermo Fisher, Karlsruhe, Germany) for 15 min, washed three times with PBS and infected with P. aeruginosa for 4 h in the presence of the plasma membrane impermeable nucleic acid stain SYTOX™ green (200 nM, Thermo Fisher, Karlsruhe, Germany). The fluorescence intensity was measured every 30 min (480/520-nm excitation/emission wavelength for SYTOX™ green and 638/686-nm excitation/emission wavelength for NucRed™ Live 647) using the Lionheart (FX) Automated Microscope system. The fluorescence signals were quantified by applying an automated primary mask recognizing the green fluorescent cells.

T3M-4 human pancreatic adenocarcinoma cells (RCB1021) were provided by the RIKEN BioResource Research Center (Ibaraki, Japan) and were cultured in DMEM/Ham’s F-12 medium containing 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO₂. The uptake of BPA into T3M-4 cells was performed with several modifications to the previously reported method [29 (link)]. Briefly, T3M-4 cells were cultured in a glass bottom dish 35 mm (Matsunami Glass Ind., Osaka, Japan) 2 days before the experiment. After washing three times with Na⁺-free Hank’s balanced salt solution (HBSS: 125 mM choline chloride, 25 mM HEPES, 4.8 mM KCl, 5.6 mM D-glucose, 1.3 mM CaCl₂, 1.2 mM MgSO₄, and 1.2 mM KH₂PO₄), 1.5 mL of BPA (1.0 mM in HBSS) was added and incubated at 37 °C for 30 min. For the BPA-absent group, only HBSS was added and incubated. After washing three times with HBSS, 1.5 mL of BITQ (10 μM in 0.5% DMSO/HBSS) was added and incubated for 5 min at RT. For nuclei staining, cells were incubated with NucRed Live647 (Thermo Fisher Scientific, Tokyo, Japan, 1:15 dilution in HBSS) at 37 °C for 30 min. Fluorescence images were acquired with a BZ-X810 (Keyence, Osaka, Japan) instrument using a DAPI-V filter cube (Keyence OP-88359, Ex: 395/25 nm, Em: 460/50 nm) and a Cy5 filter (Keyence OP-87766, Ex: 620/60 nm, Em: 700/75 nm) for BITQ and NucRed Liver647, respectively.

To capture time-lapse images of live HiDEP undergoing enucleation, Celldiscoverer 7 (Zeiss) was used. Cells were stained with Hoechst 33342 or NucRed Live 647 (ThermoFisher Scientific) to distinguish the nuclei. Thirty thousand cells per well were plated and indicated chemical compounds were added. Images were taken every 16 min for 48 h with ×20 magnification.