Cytocalcein 450

After HHP treatment, 1 × 10⁵ cells per sample were washed with PBS, resuspended in fresh DMEM/ 10% FBS, and seeded in a 35-mm glass-bottom dish (Matsunami Glass Ind., Ltd., Osaka, Japan), after which they were stored for 30–60 min at 37 °C in a 5% CO₂ incubator to encourage attachment to the dish bottom. For apoptosis/necrosis observation, the medium was gently discarded, and cells were washed 2 times with 200 μl of Assay Buffer. The cells attached to each glass-bottom dish were then incubated with a mixture of 200 μl Assay Buffer, 2 μl Apopxin Green Indicator, 1 μl 7-AAD and 1 μl CytoCalcein 450 (ab176749; Abcam) at RT for 30–60 min. For imaging ROS production, 1 ml of 5 μM MitoSOX reagent working solution (Molecular Probes) was administered to each glass-bottom dish, which was then incubated at 37 °C for 10 min. After gently washing 3 times with PBS, those cells were counterstained with Hoechst 33342 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and incubated at RT for 15 min. Finally, images were acquired using a confocal laser scanning microscope (Fluoview FV3000; Olympus Co., Tokyo, Japan).

Briefly, 2–3 × 10⁵ cells per sample were collected and washed 2 times with PBS. For the detection of PtdSer membrane translocation, MSCs were then resuspended in a mixture of 200 µl of Assay Buffer, 2 µl of Apopxin Green Indicator, 1 µl of 7-AAD and 1 µl of CytoCalcein 450 (Apoptosis/Necrosis Detection Kit; #ab176749; Abcam Co., Ltd.) as manual instructions. The samples were then kept protected from light at room temperature (RT) for 30–60 min, and 2 × 10⁴ cells were subsequently archived for flow cytometry on a BD FACSCanto II (BD Biosciences, San Jose, CA, USA) using the Flowjo software program (Flowjo LLC, BD Biosciences). The amounts of early apoMSCs (Apopxin⁺/7-AAD⁻) were analyzed and compared.
In addition, after HHP treatment, 1 ml of medium containing approximately 2–3 × 10⁵ cells per sample was mixed with 1 µl of CellEvent Caspase-3/7 Green Detection Reagent (CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit; Thermo Fisher Scientific, Inc.). The mixture was then vortexed and incubated for 60 min at RT, protected from light. In the final step, 1 µl of 1 mM SYTOX AADvanced dead cell stain solution (CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit; Thermo Fisher Scientific Inc.) was added, and then 2 × 10⁴ cells were archived for analysis on a BD FACSCanto II (BD Biosciences) using the Flowjo software program (Flowjo LLC, BD Biosciences).