Colorometric dc protein assay kit

Mice were euthanized by decapitation while under isoflurane anesthetic. Brain (hippocampal and cortical) tissues were then removed. Mitochondria derived from transfused and control mice were isolated using the commercial isolation kits (110,170, Abcam, Cambridge, MA, USA) as reported previously [21 (link)]. Briefly, hippocampal and cortical tissues were washed twice with 1 × PBS and then homogenized in an isolation buffer. Homogenates were centrifuged at 1000 × g for 5 min at 4 °C. Supernatants were centrifuged at 10,000 × g for 20 min at 4 °C. Pellets were resuspended with PBS. Furthermore, the protein concentrations of samples were measured utilizing a colorometric DC protein assay kit (Bio-Rad, Hercules, CA, USA) as described previously [22 (link)].

The activity of cytochrome c oxidase (COX) and citrate synthase (CS) was assessed spectrophotometrically (Ultrospec 2100 pro; GE Healthcare) using ScienCell assay kits (8278 and 8318, ScienCell Research Laboratory, California, USA) on isolated mitochondria derived from control and 3xTg astroglia. The activity of COX and CS was determined spectrophotometrically by measuring absorbance at 550 nm, 412 nm, respectively.
All relative quantification for enzyme assays were analyzed based on densitometry values normalized to total protein level. Protein concentrations were measured utilizing a colorometric DC protein assay kit (BioRad, Hercules, California, USA).