Bz x800 analyzer

Keratinocytes were immunostained as described above using anti-YAP and Alexa antibodies. After observation using a BZ-X810 microscope (KEYENCE), YAP-derived intensity localized in nuclei was analyzed using a BZ-X800 analyzer (KEYENCE).

A549 cells seeded in 96-well plates were transfected with one of the experimental siRNAs. After 48 h, the cells were infected with rMuV/AcGFP at an MOI of 0.05. At 96 hpi, the AcGFP brightness was analyzed using a BZ-X800 analyzer (Keyence Co., Osaka, Japan).

Newly synthesized proteins were detected using Click-iT HPG Alexa Fluor Protein Synthesis Assay Kits (C10428, Life Technologies). Approximately 25,000 BT474 cells and 30,000 MDA-MB-361 cells were seeded into 96-well plates with 100 μL RPMI medium, and allowed to adhere to the plates for 24 h. The cells were treated with fulvestrant at 100 nM, lapatinib at 100 nM, ipatasertib at 1000 nM, the double-combinations, or the triple-combination for 24 h, and then Click-iT HPG (l-homopropargylglycine), an amino acid analog of methionine containing an alkyne moiety, was incorporated into drug-treated cells for 15 h. The Click-iT HPG Alexa Fluor Protein Synthesis Assay was performed according to the manufacturer’s protocol. Fluorescent images were captured and quantified by BZ-X800 Analyzer (Keyence Corporation).

RAW-D cells (6.8 × 10³/mL) were seeded on experimental and control groups. AB and CO₃Ap were ground to a particle size of 200 μm or less and added at a concentration of 50 ng/mL RANKL for 4 days. The number of multinucleated TRAP-positive cells was counted, converted per well, and the average was compared with the control. The samples were observed with light microscopy (BZ-X800, Keyence, Osaka, Japan). The number of TRAP-positive cells was calculated using cell count of BZ-X800 Analyzer software version 1.1.1.8 (Keyence, Osaka, Japan).

Peritoneal macrophages were obtained from peritoneal lavage of WKY rats by intraperitoneal injection of 50 mL sterile saline. The abdomen was gently massaged before the lavage collection. The lavage fluid was centrifuged at 400 × g for 30 min. Mononuclear cells in the pellet were isolated using Histopaque-1083 (Sigma-Aldrich, St. Louis, MO) and transferred to culture plates. After overnight incubation at 37 °C in a 5% CO₂ atmosphere, floating cells were removed from the culture plates. Then, DiD-labeled ASCs were added to the macrophage culture plate at a 1:50 ratio. Fluorescence and phase-contrast images were captured every 5.5 min using a BZ-X800 incubator microscope system (Keyence, Tokyo, Japan) and analyzed with a BZ-X800 analyzer (Keyence, Tokyo, Japan).

To evaluate HS and CS GAG chains, HT22 cells were stained with either monoclonal anti-HS antibody 10E4 (F58-10E4 clone, 1:250 dilution; AMS Biotechnology Ltd. Abingdon, UK, RRID:AB_10891554) or monoclonal anti-CS antibody CS-56 (anti-6S, 2S, 4S antibody) (1:100 dilution; Sigma-Aldrich Corporation, St. Louis, MO, USA, RRID:AB_476879). HT22 cells (2.0 × 10³/cm²) were grown on 24-well plate and treated with sodium chlorate. The cells were fixed with 4% paraformaldehyde for 15 min and then non-specific binding was blocked with 2.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Subsequently, HT22 cells were incubated in primary antibodies with 1% BSA or immunoenhancing reagent (Can Get Signal^® Immunostain Solution B; TOYOBO, Osaka, Japan, Cat# NKB-401) overnight at 4 °C. HT22 cells were washed with PBS and immersed in 2.5% BSA or Can Get Signal^® Immunostain Solution B (TOYOBO, Osaka, Japan) containing anti-IgM-FITC (fluorescein isothiocyanate, 1:250 dilution; Jackson ImmunoResearch, PA, USA) and then incubated with 10 µg/mL Hoechst 33258 (Molecular Probes, OR, USA) for nuclei staining. After washing with PBS, fluorescence images were captured with a fluorescence digital microscope (BZ-X800, Keyence, Osaka, Japan). The fluorescent intensity was quantified using Keyence image measurement and the analyzing software (BZ-X800 Analyzer, Keyence Corporation, Osaka, Japan).

Epididymal and inguinal subcutaneous WAT was fixed in 10% formalin neutral buffer (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) for 24 h at 4 °C and embedded in paraffin following the standard protocol. Sections (5 μm) were stained with hematoxylin and eosin for 10 and 5 min at room temperature, respectively, and at least 750 adipocytes in eWAT from each mouse were visualized using a BZ-X810 microscope (Keyence, Osaka, Japan) and enumerated using a hybrid cell count software (BZ-X800 Analyzer, ver. 1.1.2.4, Keyence).