Pierce microplate bca protein assay kit reducing agent compatible kit

For analysis using the rat blood, all samples were utilized and the main focus was on the blood cytosol which refers to the extracted cytosolic components of all cells in the blood. Both isolation and purity were established as previously described (10 (link)). Cytosol/Particulate Rapid Separation Kit (BioVision Inc., CA, USA) was used to isolate the blood cytosolic components. For analysis using the different brain regions (hippocampus, pineal gland, hypothalamus, cerebrum, and cerebellum), only control and 12 h post-treatment with PDL-BA were used for analyses. Each brain part was further cut into 1–2 mm length tissue samples and placed in a sterile microtube containing N-PER^TM Neuronal Protein Extraction Reagent (Thermo Scientific, CA, USA) prior to homogenization using a handheld motorized homogenizer. For both processed blood and brain samples, Pierce^® Detergent Removal Spin Columns (Thermo Scientific) and Pierce^® Microplate BCA Protein Assay Kit-Reducing Agent Compatible Kit (Thermo Scientific) were used to purify and standardize blood cytosol samples prior to downstream analyses. All kits were used according to manufacturer’s recommendation.

Secreted inflammation-related signals quantified include IL-8 and IL-6 cytokines, whereas secreted stress-related signals measured include total heme and hydrogen peroxide (H₂O₂). Additionally, caspase-1 (CASP1) and caspase-3 (CASP3) were likewise quantified to elucidate whether detected inflammation- and stress-related signals resulted in cell death. Pierce^® Microplate BCA Protein Assay Kit-Reducing Agent Compatible Kit (Thermo Scientific, Carlsbad, CA, USA) was used to standardize cell-free supernatant prior to downstream analyses. Secreted IL-8 and IL-6 amounts in the cell-free supernatants were measured using commercially available IL-8 and IL-6 ELISA kits (R&D Systems, Minneapolis, MN, USA). QuantiChrom^TM Heme Assay Kit (BioAssay Systems, Hayward, CA, USA) was used to measure secreted heme (free heme and heme-proteins). Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences, Plymouth Meeting, Pennsylvania, PA, USA) was used to measure secreted H₂O₂. Secreted CASP1 and CASP3 were quantified usingCaspase-1/ICE and Caspase-3/CPP32 Colorimetric Assay Kits (Biovision, Milpitas, CA, USA), respectively. All kits were used following the manufacturer’s recommendations.