Cytosol particulate rapid separation 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.

The method for cytosolic/particulate separation was based on the protocol described in the Cytosol/Particulate Rapid Separation Kit (BioVision, Milpitas, CA, USA). Briefly, at the end of the designated treatments, both the medium and the adherent cells were harvested into 15 ml tubes, and the samples were centrifuged at 600 × g for 5 min. The pellets were resuspended in 40 μl of cell suspension buffer and mixed with 40 μl of cytosol releasing buffer, including 1 mM PMSF. The cell suspension was carefully layered upon 500 μl of an oil layer with 40 μl of a particulate layer at the bottom on ice for 30 s. After centrifugation at 1400 × g for 1 min, the cytosol was separated from the membrane particles by the oil layer and were transferred to new tubes. A BCA total protein assay kit (Biomiga) was used to determine the protein concentration.