Caspase 3 assay kit

The mitochondrial transmembrane potential was also measured using fluorescent cationic dye rhodamine 123 as described [26 (link)]. The fluorescence signals of the dye were measured immediately at an excitation wavelength of 488 nm and an emission wavelength of 510 nm using a fluorescence microplate reader. The results were expressed as percentage of an increase or decrease in fluorescence above the control fluorescence. Caspase 3 activity was measured using abcam Caspase 3 assay kit. One million cells were isolated using cell lysis buffer and the liquid fraction was used to analyze the caspase 3 activity as manufacturer’s instructions. The sample absorbance was measured at 450 nm and the graph was plotted using absorbance value.

In total, 5 × 10⁴ cells were seeded in a 10-cm dish and allowed to grow for 24 h. Media was removed and replaced with either serum-free DMEM or complete DMEM. Cells were collected after 24 h. Caspase activity was determined using the Caspase-3 Assay Kit (Colorimetric) (Abcam ab39401, Cambridge, UK).

For a Caspase-3 Assay Kit (ab39401, Abcam), infarct tissue was immersed in 400 μl of lysis buffer and disrupted using a homogenizer. To measure mitochondrial enzyme activities, we used the Complex II Enzyme Activity Microplate Assay Kit (ab109908, Abcam) and Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911, Abcam).

Following the usual treatment period stated above, both floating and adherent cells from vehicle‐treated control and different treatment groups were collected and subjected to Annexin V and propidium iodide staining using ‘Dead Cell Apoptosis Kit’ (Thermo Fisher, Eugene, OR, USA; Cat# V13242). As a positive control of apoptosis, the cells were treated with 20 μmol/L cisplatin (Abcam, Cambridge, UK; Cat# ab141398). Upon staining, the subsequent steps related to flow cytometry was performed as described before.²⁰ The caspase‐3 enzyme activity was measured using the caspase‐3 assay kit (Abcam; Cat# ab39401) following the supplier provided protocol. The cell lysates from control and different treatment groups were incubated with DEVD‐p‐NA substrate containing buffer for 3 hours, and absorbance at 405 nm was measured by using a Tecan Infinite® 200 PRO microplate reader.

Both MSCs LYS and MSCs CM were tested in vitro for their anti-proliferative activity on MSTO-211H, NCI-H2452 and NCI-H2052 cells in 96 multi-well plates (Sarstedt, Nümbrecht, Germany), as previously described [23 (link),24 (link)]. Briefly, 1:2 serial dilutions MSCs LYS and MSCs CM were performed in 100 µL of culture medium/well, and then, 10³ tumor cells were added to each well. Cell growth was evaluated after 7 days of culture by measuring the optical density at 550 nm in an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazoliumbromide) assay [25 (link)]. Cell death was assessed by Hoechst 33342 and propidium iodide dual staining and by using the Apoptosis/Necrosis Detection Kit (Abcam, Cambridge, UK). Caspase-3 activity was measured by the Caspase-3 Assay Kit (Abcam) following the supplier’s protocols. The distribution of the cells in the cell cycle (determined by PI staining and flow cytometry analysis) was determined as described elsewhere [26 (link)].