The first strand cDNA was created according to the manufacturer protocol, which then synthesized the second strand cDNA.
Cell viability was evaluated using the AlamarBlue™ reagent (AbD Serotec, Kidlington, UK), according to the manufacturer protocol.
Samples were run on a 4 12% Nu-PAGE gel (Life technologies), transferred to Hybond-C PVDF membrane according to the manufacturer protocol (Amersham Biosciences, GE Healthcare).
DNA was extracted using the Sherlock AX Kit (A&A Biotechnology, Poland) following manufacturer protocol.
SP concentrations in samples of plasma were measured by ELISA, following the manufacturer protocol (Abcam, Cambridge, MA).
Protein concentration in the tissue lysates was measured using BCA Protein Assay Kit Piercee, Rockford, IL) according to manufacturer protocol.
Total RNA was isolated from 60 frozen/ fresh samples using Trizol (Invitrogen, Life Technologies, California, USA) according to manufacturer protocol.
Wizaed SvGel and PCR Clean Up System (Promega, US) was used to clean PCR products following manufacturer protocol.
following manufacturer protocol.
Each sample was analyzed in duplicate according to manufacturer protocol.
Neuro-2a cells were transfected using Amaxa nucleaofection device following manufacturer protocol.
