Labeling living/dead MC3T3-E1 cells by FDA/PI double staining. Labeling of cytoskeleton proteins and nuclei by FITC (UE, China)/DAPI (Thermo fisher, USA). FDA (Thermo fisher, USA) labeled living cells turned green, while PI (Thermo fisher, USA) labeled living cells turned red. Observe by fluorescence microscope on day 1, 3 and 7. Among the high-rate images of cytoskeleton staining, select 50 cells randomly from each group on day 1, 3 and 7. Measure the angle between the cell long axis and the actin microfilament direction and the ratio of the cell long axis to the wide axis, and the data were processed and compared.
Detect cell vitality on day 1, 3 and 7 by Alamarblue working solution (Thermo fisher, USA; volume ratio of α-MEM of 10 % FCS: Alamarblue = 9:1). Add 150 μL to each well and incubated in cell incubator for 2 h. Then take 100uL samples from each well and placed in a 96-well plate, and measure optical density (OD) at 570 nm and 600 nm by enzyme labeling instrument (Bio-Rad, USA).
Detect ALP activity of cells on day 1, 3 and 7. The cells were lysed with enhanced RIPA lysate (BOSTER, China), and the protein concentration of the corresponding samples was detected by BCA kit (Thermo fisher, USA) after centrifugation. The ALP activity of cells was determined by nitrobenzene phosphate assay, and the ALP activity was normalized. The unit was expressed as NP/ug/min.
Detect cell vitality on day 1, 3 and 7 by Alamarblue working solution (Thermo fisher, USA; volume ratio of α-MEM of 10 % FCS: Alamarblue = 9:1). Add 150 μL to each well and incubated in cell incubator for 2 h. Then take 100uL samples from each well and placed in a 96-well plate, and measure optical density (OD) at 570 nm and 600 nm by enzyme labeling instrument (Bio-Rad, USA).
Detect ALP activity of cells on day 1, 3 and 7. The cells were lysed with enhanced RIPA lysate (BOSTER, China), and the protein concentration of the corresponding samples was detected by BCA kit (Thermo fisher, USA) after centrifugation. The ALP activity of cells was determined by nitrobenzene phosphate assay, and the ALP activity was normalized. The unit was expressed as NP/ug/min.