Enzyme standard instrument

Cells were seeded in 96-well plates at 1 × 10⁴ cells per well in 10% FBS-supplemented 1640. The following day, the cells were treated with 0.15, 0.5, 1.5, 5, 15, and 50 μM Dox and 5-FU for 48 h. Then, cell growth was evaluated using an MTT cell viability assay system according to the manufacturer's protocol. Following the colorimetric reaction, the optical density was determined at 490 nm using Enzyme Standard Instrument (BioRad, USA) [21 (link)].

The rats were sacrificed immediately after tDCS treatment on POD 3 as described in the TTC staining part. Blood samples (5 ml) were collected from the rat heart. Then, the CIP tissue of the brain was immediately separated and frozen. The serum was collected from blood after centrifugation at 1500 rpm for 15 min at 4 °C. The CIP samples were weighed and homogenized in radioimmunoprecipitation assay lysis buffer supplemented with the proteinase inhibitor phenylmethylsulfonyl fluoride using a homogenizer device (Leica, Heidelberg, Germany). The supernatant was acquired after centrifugation at 12,000 rpm for 15 min at 4 °C. The serum was used for NSE enzyme-linked immunosorbent assay (ELISA) detection (Elabscience, China), and supernatant of CIP was used for inflammatory cytokines ELISA detection (i.e. IL-6, IL-1β, IL-10, TNF-α; ImmunoWay, USA). Experiments were performed according to the manufacturer’s instructions and the absorbance was measured by using an enzyme standard instrument at 450 nm (Bio-Rad, USA).

Following varying treatments, 10 μL CCK-8 at 0 h, 24 h, 48 h, and 72 h was added to the AS-BMSCs for 1-h culturing in accordance with the CCK-8 kit instructions (C0037, Beyotime). The absorbance value was tested utilizing an enzyme standard instrument (Bio-Rad Laboratories, Hercules, CA, USA) at 450 nm. A growth curve was constructed based on the absorbance values in order to determine cell viability.