Biorevo bz 9000

Cells were cultured in medium supplemented with Alexa-488-labeled LDL (Invitrogen) for 1 hour and washed twice with PBS. Then, nuclear staining was performed using 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific), followed by staining with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical). After fixation with a fluorescent microscope (BIOREVO BZ-9000, Keyence), the samples were observed under a fluorescence microscope (BIOREVO BZ-9000, Keyence).

For transwell migration assays, 2×10⁴ cells were placed in the top chamber on a non-coated membrane (24-well insert; pore size, 8 μm; BD Bioscience, San Jose, CA, USA). For invasion assays, 2×10⁴ cells were placed in the top chamber on a Matrigel-coated membrane (24-well insert; pore size, 8 μm; BD Bioscience). In both assays, the cells were placed in serum free medium, and medium supplemented with 20% FBS was used in the lower chamber. The cells were incubated for 48 h, and those that did not migrate or invade through the pores were removed with a cotton swab. The cells were fixed with methanol, stained with DAPI (Dojindo Laboratories, Kumamoto, Japan), and then counted using a BZ-9000 BioRevo (KEYENCE, Elmwood Park, NJ, USA) and ImageJ software (open resource software obtained from https://imagej.nih.gov/ij/).

Phosphorylation of the histone H2A variant (γ-H2AX) is a marker of DNA double-strand breaks, which is the gravest form of DNA damage [21] (link). We investigated the DNA double-strand breaks induced during t-PDT using the OxiSelect DNA Double-Strand Break Staining Kit (Cell Biolabs, Inc.). Cells (TE-11R) were treated with the indicated talaporfin sodium with or without subsequent irradiation, and were stained with an anti-phospho-histone antibody 24 h after treatment. DNA double-strand breaks labeled with FITC-conjugated secondary antibody were assessed using a fluorescence microscope (BZ-9000 BIOREVO, Keyence Corp., Osaka, Japan).

Tissues embedded in paraffin were sliced into sections and stained with H&E, and Sirius red using the standard protocol for histopathology analysis of liver. Cryopreserved liver tissues were sliced and used for oil red O staining with hematoxylin counterstaining. Stained slides were observed under a BZ-9000 BioRevo digital microscope (Keyence Corp., Osaka, Japan), and were analyzed using ImageJ.

To study the distribution of the fluorescein derivatives in B. subtilis cells, we used a setup based on the inverted motorized BZ-9000 BioRevo fluorescence microscope Keyence (Itasca, IL, USA.) equipped with an HC PL Apo 100x1.40 oil lens (Nikon, Tokyo, Japan), GFP filter cube (excitation filter 470/40, emission filter 525/50), and temperature control chamber. The images were processed using FIJI ImageJ distribution [37 (link),38 (link)].

HeLa cells were cultured (12 plates, mean 253,000 cells/plate) for 24hr at 37ºC (5% CO₂). Recombinant coxsackievirus B (pMKS1) expressing an enhanced green fluorescent protein (EGFP-CVB) was prepared as previously described [17 (link)]; half were exposed to NB-UVA (2000 μW/cm²) for 20min while the other half were not exposed. HeLa cells were then transfected with NB-UVA-exposed or NB-UVA-unexposed virus (multiplicity of infection (MOI) = 0.1). Coxsackievirus is considered highly lytic [18 (link)]. After 6hrs, supernatant was removed, and cells were washed twice with 1x sterile PBS (pH = 7.0). New DMEM media was added and cells were incubated at 37ºC (5% CO₂). Dead cells in the supernatant (floating cells) were collected and quantified 24hrs later using an automated cell counter (Biorad T20). Six plates (3 NB-UVA-exposed and 3 unexposed) were assessed for live cells. Of the remaining six plates, the 3 plates transfected with NB-UVA-exposed virus were exposed to an additional 20min of NB-UVA (2000 μW/cm²). After 24hrs at 37ºC (5% CO₂), imaging was performed using a BZ-9000 BioRevo (Keyence Corp., Itasca, IL). Dead and live cells were determined by the Trypan Blue 0.4% (1:1)(Gibco) method and counts were obtained using an automated cell counter (Biorad T20).