Md m5 spectramax reader

H9-ECs were cultured in 6-well plates. Caspase 3 activities were performed using Caspase 3 Assay Kit (Beyotime) according to the manufacturer’s instructions. Standard curve of pNA (p-nitroaniline) concentration relative to A405 was firstly generated. At least 1 × 10⁷ cells were collected by centrifugation at 600 g for 5 min at 4 °C. The cell pellets were washed with DPBS and re-suspended in 1× lysis buffer at a concentration of 100 μl per 2 × 10⁷ cells, incubated on ice for 15 min and then centrifuged at 16000–20000 g for 15 min at 4 °C. Concentration of proteins was measured by Bradford method. Appropriate amount of protein was put in a 96-well plate and 10 μl of Ac-DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide) (2 mM) was added per well, and then incubated for 2 h at 37 °C. Absorbance at 405 nm was read using a MD M5 SpectraMax reader (Molecular Devices).

Caspase 3 activities were performed using caspase 3 Assay Kit (Beyotime) according to the manufacturer′s instructions. H9‐CMs were digested with Trypsin‐EDTA (Gibco) and collected by centrifugation at 600 g for 5 minutes at 4°C. The cell pellets were washed with DPBS (Gibco) and re‐suspended in 1 × lysis buffer at a concentration of 100 μL per 2 million cells, incubated on ice for 15 minutes and then centrifuged at 16 000‐20 000 g for 10‐15 minutes at 4°C. Appropriate amount of protein was put in a 96‐well plate, and 10 μL of Ac‐DEVD‐pNA (acetyl‐Asp‐Glu‐Val‐Asp p‐nitroanilide) (2 mmol/L) was added per well and then incubated for 60‐120 minutes at 37°C. Absorbance at 405 nm was read using a MD M5 SpectraMax reader (Molecular Devices).

H9-ECs were cultured in 96-well plate and cell viability analyses were performed using CCK8-based in vitro cell proliferation and cytotoxicity assay kit (Beyotime) according to the manufacturer’s instructions. Cells were incubated in the presence of 10 μl CCK8 reagent per well for 3 h. Absorbance at 450 nm was measured using a MD M5 SpectraMax reader (Molecular Devices).