Varioscan flash microplate reader

Fasting venous blood samples were collected from 139 patients with ischemic stroke and 58 healthy subjects in lithium heparin sterile tubes and immediately centrifuged at 1200× g using assay kits according to the manufacturer’s instructions (Cell Biolabs, San Diego, CA, USA). Plasma samples were aliquoted and stored at −80 °C until further use. Two alternative biochemical parameters reflecting redox homeostasis such as reactive oxygen species (ROS) and oxidized glutathione (GSSG) levels were measured by Varioscan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) in all plasma samples of IS patients and healthy subjects (GSSG levels were assessed in 91 patients with IS and 44 controls), as described previously [30 (link)].

The WST-8 and lactate dehydrogenase (LDH) assays were used to assess the viability of RAW 264.7 cells. The Cell Counting Kit 8 (CCK-8) was used for the WST-8 assay and performed according to the manufacturer’s protocol (Dojindo, Kumamoto, Japan). Briefly, RAW 264.7 cells were seeded at 2.5 × 10⁴ cells/well in 96-well plates for 24 h followed by treatment with the indicated concentrations of astaxanthin or DMSO for an additional 24 h. Afterwards, media was replaced with 0.5 µg/mL CML-HSA-containing media and was incubated for 3 h. After 2 h of CML-HSA treatment, 10 µL/well CCK-8 was added for WST-8 assay and the mixture was then incubated for another 1 h. Finally, after a total of 3 h of CML-HSA treatment (Figure 1A), or as otherwise indicated in Figure 4A, absorbance at 450 nm was measured by a Varioscan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) [40 ,61 ]. The method described by Sato et al. was used to perform LDH assay [40 ]. To evaluate the effect of CML-HSA and astaxanthin on cell cytotoxicity, 50 µL of culture media from each well of the treated culture plate was collected and used for performing the LDH assay.