pLAMP1-mCherry (42) was a gift from A. Palmer (Addgene plasmid no. 45147; http://n2t.net/addgene:45147; RRID: Addgene_45147). The plasmid was transfected with the Neon electroporation system (Thermo Fisher Scientific, MA, USA) using 10-μl tips. The next day, the media was replaced with osteogenic media. The cells, either in osteogenic or normal media, were washed once with phosphate-buffered saline (PBS) and stained with LysoTracker Red DND-99, MitoTracker Deep Red FM, or Hoechst 33342 (Thermo Fisher Scientific) at 1:10,000 for 15 min at 37°C. Subsequently, cells were washed three times with PBS and Live Cell Imaging Solution (Thermo Fisher Scientific) in the presence of ProLong Live Antifade Reagent (Thermo Fisher Scientific). For mineral staining of live cells, a protocol modified from (43) was used. In short, live cells were incubated with 0.01% Alizarin Red S solution (Wako Pure Chemical Industries) in culture media for 15 min. Live-cell imaging was performed with the Leica TCS Sp8 confocal microscope (Leica Microsystems, Germany), the N-SIM super-resolution microscope (Nikon Instruments, Japan), or the SpinSR10 spinning disk confocal super-resolution microscope (Olympus Corporation, Japan), according to the manufacturer’s instruction.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



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