Polytron tissue homogenizer

Lungs were homogenized in 1 ml of saline using the Polytron Tissue Homogenizer (Kinematica, Luzern, Switzerland). Homogenates and BALs were subjected to 10-fold serial dilutions in saline and cultured in tryptic soy agar (Wisent) at 37°C and CFUs were counted 18 to 24 h later.

After exposure to radiation, the rats were kept alive for a specific amount of time and then given a lethal dose of Pentothal. Tissues from the cerebral cortex and cerebellum were then dissected out under a stereomicroscope (Nikon Eclipse CFI60). The tissue samples were placed in 500 μl of phosphate buffer (PBS; 0.015M phosphate buffer, 0.15M NaCl, pH 7.2) containing 0.1mM pepstatin A, 0.02mM N-(trans-epoxysuccinyl)-L-leucine-4 guanidinobutylamide (E-64), 1mM phenylmethanesulfonyl fluoride (PMSF) and 2mM ethylenediaminetetraacetic acid (EDTA) protease inhibitors (all from Sigma-Aldrich). The samples were disaggregated and homogenized in a Polytron tissue homogenizer (Kinematica AG, Littau, Luzern, Switzerland) at 35,000 rpm for 5 min on ice, and finally ultrasonically lysed in a Branson W-250 sonifier (Branson Ultrasonic Corporation, USA) by means of 5 10-pulse sonication cycles with a 50% duty cycle output. The whole process was performed on ice. The lysate obtained was centrifuged at 15,000 g for 10 minutes at 4°C. The supernatant was then aliquoted and frozen at −80°C until use.

Tissues were first processed with a Cuisinart food processor (Conair Corp., Stamford, CT, USA). Duplicate 1 g aliquots were weighed into centrifuge tubes and spiked with 200 µL of the working internal standard solution. Samples were then homogenized in 20 mL of acetonitrile with a Polytron Tissue Homogenizer (Kinematica, Bohemia, NY, USA). 10 mL of hexane was added, and the samples were shaken on a platform shaker for 10 min at 250 rpm. After centrifugation at 1200× g for 10 min, the hexane was removed to waste, and the acetonitrile was transferred to a 25 mL volumetric flask. Samples were brought to volume, added to 15 g of anhydrous sodium sulfate, shaken for one minute and centrifuged again at 1200× g for 10 min. A 12.5 mL aliquot of the extractant was evaporated to dryness at 60 °C with a gentle stream of nitrogen, reconstituted with 200 µL of 50% methanol and centrifuged at 12,000× g for 5 min before analysis on the HPLC system.
Plasma samples (250 µL) were spiked with 50 µL of the working internal standard solution prior to the addition of 3.5 mL of acetonitrile. After vortex mixing, the samples were centrifuged at 1200× g for 10 min. The extractant was transferred to a new tube and evaporated to dryness at 60 °C with a gentle stream of nitrogen, reconstituted with 100 µL of 50% methanol and centrifuged at 12,000× g for 5 min before analysis on the HPLC system.

Samples weighing 2 g were extracted with 10 mL of 80:20 (v/v) methanol:water with formic acid (1%) using a Polytron tissue homogenizer (Type PT 10–35; Kinematica GmbH, Luzern, Switzerland) for 1 min and centrifuged for 20 min at 4000× g [9 (link)]. The pellets were re-extracted, and supernatants were pooled and then evaporated under vacuum at room temperature using a Speedvac (SC210A; Savant Instruments, Farmingdale, NY, USA). The residues were re-suspended in 10 mL of methanol/formic acid (99:1, v/v). Aliquots were stored at −80 °C before analysis. All analyses were performed in triplicate. Using external calibration curves based on quercetin and kaempferol analytes (which are always detectable in all the samples), the content of polyphenolics in the extracted sample averaged 15 mg/kg.

Parotid gland biopsies were snap-frozen in liquid nitrogen after surgery and cryo-preserved at −80°C until use. RNA was isolated from parotid gland samples using a polytron tissue homogenizer (Kinematica AG, Littau-Lucerne, Switzerland) in the presence of STAT60 RNA reagent (Tel-test Inc., Friendswood, TX, USA) according to the manufacturer’s protocol. After isolation RNA was purified using the RNeasy Mini System [Clean-up-protocol (#74106, Qiagen, Venlo, the Netherlands)]. RNA quality was checked using the Bioanalyzer 2100 system (Agilent) and quantified using the Qubit1.0-platform (#Q32857, Invitrogen Life Technologies, Breda, the Netherlands). cDNA was synthesized using Superscript RT-III and oligo-dT primers according to the manufacturer’s protocol (#18080-051, Invitrogen).