Sod assay kit

An SOD assay kit (BioVision, Inc., Milpitas, CA, USA) was used to measure SOD activity.

SOD activity, glutathione peroxidase (GPx) activity, lipid peroxidation of MDA, and LPO in plasma were measured by using SOD Assay Kit (ab65354), Glutathione Assay Kit (ab102530), MDA Assay Kit (ab118970), and LPO Assay Kit (ab133085), respectively, according to the manufacturer’s instructions (Abcam, Cambridge, MA, USA). The optical density for SOD, GPx, MDA, and LPO kits was read at 450, 340, 532, and 500 nm wavelengths, respectively, using an Epoch microplate spectrophotometer (BioTek^® Instruments, Inc. Highland Park, VT, USA). The intra-assay CV for SOD, GPx, MDA, and LPO was 2.9%, 0.1%, 1.3%, and 1.1%, respectively. The inter-assay CV for SOD and GPx was 2.7% and 0.1%, respectively.

SOD activity was determined using SOD assay kit (Abcam USA, Cat No. ab65354) according to the manufacturer’s instruction. 20 µl/well supernatant of different groups in triplicate was added followed by the addition of 200 µl of WST solution in each well included blank 1, 2 and 3. Then, added 20 µl of dilution buffer in blank 2 and 3. Enzyme working solution (20 µl) was added to each sample well and blank 1. Gently shake the plate for thoroughly mixing and incubated for 37°C for 20 minutes. Absorbance was measured at 450 nm using a microtiter plate reader.
Following equation was used for calculating SOD activity (% inhibition rate)

The blood samples collected from the rats were centrifuged for 10 min at 1,000 × g and 4°C to harvest the supernatant. Myocardial tissues in the I/R region of rats were homogenized on ice. The homogenate was then centrifuged for 10 min at 1,000 × g and 4°C, and the supernatant was collected. The H9c2 cells of each group were fostered for 48 h in a relevant medium. The cells were collected and lysed for 30 min in the cell lysate (Beyotime, Shanghai, China) on ice, followed by centrifugation for 10 min at 1,000 × g and 4°C. The supernatant was then collected. The iron concentration in the supernatant samples was quantified using the Iron Assay Kit (ab83366; Abcam, Cambridge, United Kingdom). Furthermore, the levels of ROS, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in the supernatant samples of myocardial tissues and H9c2 cells were assessed using the ROS Assay Kit (KA3842; AmyJet Scientific, Wuhan, China), MDA Assay Kit (ab238537; Abcam, Cambridge, United Kingdom), SOD Assay Kit (ab65354; Abcam, Cambridge, United Kingdom), and GSH Assay Kit (ab112132; Abcam, Cambridge, United Kingdom). The detection procedure was followed strictly according to the manufacturers’ instructions.

The levels of malondialdehyde (MDA), and activities of superoxide dismutase (SOD), catalase (CAT), and caspase‐3 were measured using the MDA assay kit (Abcam), SOD assay kit (Abcam), catalase assay kit (Abcam), and caspase‐3 assay kit (Abcam), respectively, according to the manufacturer's protocol.
5′‐GGA TGC CTT TGT GGA ACT GTA TT‐3′ (sense) and
3′‐TAC AGT TCC ACA AAG GCA TCC‐5–5′ (antisense).
5′‐GGA TGC CTT TGT GGA ACT GTA TT‐3′ (sense) and
3′‐TAC AGT TCC ACA AAG GCA TCC‐5′ (antisense).

An amount of 2 × 10⁶ cells/well were cultured in 96-well plates, and after 24 h were treated with ALAD-PDT. SOD activity was determined immediately after the treatment (45′) and at different time points after the treatment, namely, 30 min (45′ + 30′) and 1 h (45′ + 1 h), using a SOD assay kit (Abcam, Cat No. ab65354) according to the manufacturer’s instruction. After the preparation of samples, 20 µL of enzyme working solution was added to each well. After incubation of 20 min at 37 °C, the absorbance was measured at 450 nm using a microplate reader (Synergy H1 Hybrid BioTek Instruments). SOD activity was calculated as a percentage of inhibition rate.