The amount of RNA extracted from tissue specimens was measured using the Quant-iT Ribo Green RNA assay (Invitrogen), adhering to the manufacturer's instructions, and measuring fluorescence for 0.1 seconds at excitations and emissions of 485 nm and 535 nm, respectively, using a Victor3 V 1420 Multilabel Counter (Perkin Elmer, Waltham, MA).
We explicitly deemphasized reaction time in the instructions and measured performance by the percent correct trials (and their derivate false positives and hits), from the total of 96 trials comprising each task.
Cell apoptosis was analyzed using the Annexin V-FITC/PI Apoptosis Kit (Biosea, Beijing, China) according to the instructions and measured by flow cytometry.
Assays were performed according to manufacturer's instructions, and measured in Tecan GENios pro microplate spectrofluorometer (Tecan Group, Männedorf, Switzerland) with the Magellan V5.03 system (Tecan Group).
After 6 days incubation at 37°C and 95% humidity in 5% CO2 atmosphere, intracellular ATP was extracted using the CellTiter-GIo Luminescent Kit (Promega, USA) according to the manufacturer's instructions and measured by microplate luminometer (Berthod Diagnostic system, Germany).
For apoptosis detection after 48 h treatment, cells were stained with AnnexinV-APC/7-AAD (BD Pharmingen, Uppsala, Sweden) according to the manufacturer's instructions and measured by flow cytometry.
The adherent and floating cells were combined and treated according to the manufacturer's instruction and measured with FITC/PI staining using flow cytometry (Becton Dickinson, San Jose, CA).
On day 5, the stimulated cells were labeled by Brdu solution followed by developing the Eu-fluorescence as the manufacturer's instruction and measured the Eu-fluorescence counts using Victor2 1420 multilabel counter (PerkinElmer Life Sciences, Turku, Finland).
Angelerio's instructions and measures facilitated interventions and changed the way in which local health officers were selected.
Proliferation was quantified by CellTiter-Glo® Luminescent Cell Viability Assay and apoptosis was determined by CaspaseGlo® 3/7 Assay (both Promega) according to the manufacturer's instructions and by measuring luminescence with an Orion II luminometer (Berthold Detection Systems, Pforzheim, Germany).
A non-stretchable paper measuring tape (range 0 135), which was especially produced for the study, and measuring instructions for use were sent to all participants along with the questionnaire.
