24 wells

We performed short-term (0 h) and long-term (24 and 48 h) viability experiments on cells collected from the target outlet. The PE Annexin V Apoptosis Kit I (BD Biosciences, United States) was used to detect short-term cell viability. The kit contains two dyes, PE Annexin V and 7-Amino-Actinomycin (7-AAD), which can detect early apoptotic cells (PE Annexin positive, 7-AAD negative), late apoptotic cells or dead cells (PE Annexin positive and 7-AAD positive), and viable cells (PE Annexin and 7-AAD negative). Cell viability was calculated as the ratio of the number of viable cells to the total number of cells. For the long-term cell viability, the collected cells were cultured in 24 wells (Corning, United States) for further observation of the cell viability and morphology after going through the chip at 24 and 48 h. All cell viability tests were designed with the control groups.

The function of osteoclasts was assessed by the resorption assay. Briefly, BMMs were inoculated in osteo assay surface plates containing 24-wells (Corning, USA) at a concentration of 10 × 10⁴/well. Then groups of BMMs were induced to differentiate toward osteoclasts as before. After 6 days, trypsin digestion and three PBS washes were performed on the cells. Following that, images were captured with a simple light microscope (Zeiss), and Image Pro-Plus (Version-6.0) was employed to analyze the region of the resorption depression.