Cell culture and osteogenic differentiation
Live-cell imaging
Western blot analysis
High-resolution SE-ADM
SE-ADM culture dish holder
Image processing
Calculation of particle diameters
Immunofluorescence staining of the cells and observation by optical microscopy
Immunolabeling of osteoblasts using antibody and 60-nm gold colloids
EDX spectrometric analysis
Raman microscopy
Transfection of CRISPR-Cas9 ribonucleoprotein complex
Measurement of supernatant alkaline phosphatase activity
Quantitative reverse transcription polymerase chain reaction
Genomic PCR and subcloning for sequencing analysis
Inhibitor treatment
The RNP transfected cells in six-well plates were harvested with trypsin-EDTA and resuspended in 2% fluorescence correlation spectroscopy containing PBS. Singlet populations were then sorted into 96-well plates with an SH800Z cell sorter (Sony Imaging Products & Solutions Inc., Japan) in a single-cell sorting mode. Recovery rates of each single cell were around 80% when a 130-μm disposable chip (Sony Imaging Products & Solutions Inc.) was used. The sorted cells that formed a visible single colony in each well within a week became ready to be passaged 2 to 3 weeks after sorting.