Eclipsenet

Monodansylcadaverine (MDC), which is an autofluorescent compound due to the dansyl residue conjugated to cadaverine, has been shown to accumulate in acidic autophagic vacuoles. The concentration of MDC in an autophagic vacuole is the consequence of an ion-trapping mechanism and an interaction with lipids in autophagic vacuoles (autophagic vacuoles are rich in membrane lipids). The use of MDC staining is a rapid and convenient approach by which to assay autophagy, as shown in cultured cells [15 (link)]. Autophagic vacuoles were labeled with MDC by incubating cells on coverslips with 0.05 mM MDC in PBS at 37 °C for 10 min. After incubation, the cells were washed four times with PBS and immediately analyzed by fluorescence microscopy (Nikon Eclipse E 200, Japan) equipped with a blue filter. Images were obtained with a Nikon Digital Sight DS-SM (Nikon, Japan) camera and processed using the program EclipseNet, version 1.20.0 (Nikon, Japan).

RAW 264.7 cells (American Type Culture Collection [ATCC], Manassas, VA, USA), a murine monocytic cell line that differentiates in the presence of RANKL, was used for measurements of osteoclastogenesis and osteoclast activity. Cells were amplified on nonadherent culture substratum in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and penicillin-streptomycin in a 37 °C, 5 % CO₂, humidified incubator and were passaged by manual dissociation. Cells were seeded on 16-well osteologic slides (BD BioCoat; BD Biosciences Discovery Labware, Billerica, MA, USA) at 0.125 × 10⁴ cells/0.25 ml/well in triplicate and were allowed to adhere for 24 h, then stimulated to differentiate (30 mg/ml RANKL; PeproTech, Rocky Hill, NJ, USA) with or without drug for 7 days. Plates were subsequently rinsed, bleached, dried, and analyzed for matrix degradation using a Nikon COOLSCOPE (Nikon Instruments, Melville, NY, USA). Matrix resorption was quantified using EclipseNet (Nikon Instruments)/Visiopharm (Visiopharm, Hørsholm, Denmark) software (see Additional file 1).