Housing conditions of experimental animals
Male SD rats were used for the animal experiments. All animals used were housed in polypropylene cages (3-4 animals per cage) with sterilized straw. Hydration and nutrition were provided ad libitum . Temperature of the housing complex was always kept around 25 ºC±3 ºC with relative humidity at 50-55%. Animals used for a particular study were always from the same cohort. Light/dark cycles were of 12 h each. All care was taken to avoid any discomfort or pain during experimentation. All studies were carried out in accordance with the Institutional Animal Ethics Committee’s regulations which are based on the NIH Guidelines for care and use of animals during experimental procedures.
Collection of tissues and blood samples and extraction of plasma in Rats
The site of blood collection was the retro-orbital vein. Rats underwent general anesthesia using Ketamine:Xylazine recommended dose (i.p.) prior to blood extraction. The blood was collected in EDTA/citrate lined tubes. These tubes were then centrifuged for 15 min at 3500 rpm. PI cocktail (Cat. no. P8340, Sigma, USA) was added immediately thereafter. After the sacrifice of rats using lethal dose of ketamine:xylazine, their lungs were sectioned and rinsed in cold PBS. All plasma and lung tissue samples were snap frozen using liquid nitrogen and stored in -80oC.
Lung tissue, after de-freezing to room temperature, was homogenized in RIPA buffer (1:10 w/v) and sonicated (three times for 5 seconds each) briefly. Then the homogenate was centrifuged (15,000 g ; 20 min) at 4°C and the clear supernatant was used for assays.