Kimble dounce tissue grinder set

Frozen tissue was homogenised on dry ice using a 2 ml Kimble dounce tissue grinder set (Sigma-Aldrich, D9063), suspended in RNA lysis buffer from the Qiagen RNeasy Micro Kit (Qiagen, 74004) and passed 10 times through an 18 gauge syringe needle before proceeding with RNA isolation according to the manufacturer’s protocol including the optional DNA degradation step using the Qiagen RNase-Free DNase kit (Qiagen, 79254). RNA quality was assessed on the Agilent 2100 bioanalyser using the RNA 6000 nano kit (Agilent, 5067-1511).

Briefly, frozen tissues were placed into a KIMBLE Dounce tissue grinder set (Sigma) with 10 volumes of ice-cold phosphate buffered solution and then centrifuged at 5500 rpm for 15 min (4 °C). Then, the supernantants were used for evaluation of oxidative stress. On evaluation of oxidative stress, the following antioxidant enzymes were determined: SOD and GPx, as well as the lipid peroxidation (MDA) level. Activities of SOD and GPx and MDA levels were determined colorimetrically using kits (Merck, Darmstadt, Germany) following the manufacturer’s instructions. The results were normalized to the total protein content determined with the Bradford method using bovine serum albumin as standard, while MDA level was normalized as percentage of control values.
AChE activity was measured according to a previously reported protocol [87 (link)].