Cell volume measurement

MC3T3-E1 cells in the logarithmic growth phase were digested by conventional methods and prepared as a cell suspension. Cells from the experimental and control groups were separately seeded in the perfusion groove of a custom-made culture plate, and after 20 min of cell adhesion, the experiment was conducted. An inverted phase contrast microscope (DMI6000 B; Leica, Mannheim, Germany) was used in fixed view mode to continuously acquire cell images. Image-Pro Plus (Media Cybernetics, Rockville, MD, USA) was used to control image acquisition. The perfusion speed was set at 4 ml/min. Cells were soaked in isotonic solution for 5 min, and the baseline cell volume was observed. The cells were subsequently perfused with Hypo for 20 min and subsequently re-perfused with isotonic solution for 5 min. Subsequent to the experiment, Image-Pro Plus was used to analyze and measure cell volume. Standardized cell volume (Vst) was calculated according to the formula Vst = (Vt/Vo) × 100, where Vt was the cell volume measured in real time and Vo was the average volume of the same cell under isotonic conditions. All the experiments were performed at room temperature (20–24°C).