Nbt bcip staining kit

Transfected hDPSCs were planted in a 12-well plate and grown for 7 days in mineralization induction medium. The quantitative analysis of ALP activity was performed using an alkaline phosphatase detection kit (Jiancheng, Nanjing, China) in accordance with the instructions. After 30 min of fixation with 4% paraformaldehyde, ALP staining was conducted with the BCIP/NBT staining kit (Beyotime, Shanghai, China).

ALP staining of MC3T3-E1 cells that had been induced for 7 days in osteogenic media was performed with the BCIP/NBT staining kit (Beyotime Biotechnology, China) following the manufacturer’s instructions. A digital camera was used to capture representative images.

Cells were cultured in an osteogenic medium (OM) for 7 days and underwent ALP staining and quantification. A BCIP/NBT staining kit (C3206, acquired from Beyotime Institute of Biotechnology) was used for cell staining after cell fixation. ALP activity was detected using ALP test kits (P0321M, Beyotime). Thereafter, cells were collected, centrifuged at 1000 g for 10 min, and then treated with Triton‐X100 and observed. Later, the OD was measured when the wavelength was 520 nm.

At 7 and 14 days after osteogenic induction, the transfected hDPSCs were rinsed with phosphate-buffered saline (PBS) and then fixed in 4% (w/v) paraformaldehyde. Subsequently, hDPSCs were treated with BCIP/NBT staining kit (Beyotime). Measurement of ALP activity was conducted using the ALP Activity Assay Kit (Beyotime) with absorbance being detected at 520 nm.

The ALPS was used to evaluate the activity of ALP after osteogenic differentiation of BMSCs according to the method of the BCIP/NBT staining kit (Beyotime, China, #C3206), removed the culture medium, washed it twice with precooled PBS, added 4% paraformaldehyde fixing solution, placed it at room temperature for 10 minutes, washed it twice, added substrate solution to each well, and incubated it in an incubator at 37°C for 45 minutes. During this time, it was vital to observe constantly, avoid over staining, and remove the solution. After being washed, cells were observed using a Leica Confocal 222 microscope. ImageJ software (NCI, USA) was used to analyze the images.

ALP staining was performed using a BCIP/NBT staining kit (Beyotime, China). After osteogenic induction for 7 days, cells were fixed and ALP staining was performed following the manufacturer’s instructions. Mineralized nodule formation was determined by ARS staining. After osteogenic incubation for 21 days, cells were fixed and stained with 0.1% ARS (Sigma-Aldrich, USA) for 20 min.

To examine early markers of osteogenic differentiation, alkaline phosphatase (ALP) staining and ALP activity quantification were conducted. The hBMSCs were divided into three groups: (1) PM, (2) OM and (3) OM with RBC‐apoVs. After 5 days of induction, we performed ALP staining using a BCIP/NBT Staining Kit (Beyotime Biotechnology, China). Meanwhile, we quantified the ALP activity with an ALP Activity Assay Kit (Nanjing Jiancheng, China) according to the manufacturer's protocol. The absorbance at 520 nm was detected. ALP activity normalized to the total protein content was calculated.