Next, we compare the proposed cooperative transmission protocol with the direct transmission protocol (DT protocol).
IgG anti-ApoA-1 antibodies were detected by a modification of the direct ELISA protocol described previously [ 2, 27].
The performance of our protocol is compared to the Directed Diffusion protocol.
A mouse monocolonal GFAP antibody (GFAP mAb, 25 µg, Sigma) was coupled to 1.25 million Microplex microspheres 133 (Luminex) using EDC Thermo Scientificc) and Sulfo-NHS (Thermo Scientific) as directed by the manufacturer protocol.
A rabbit polyclonal GFAP antibody (GFAP pAb, 145 µg, Dako) was coupled to biotin using EZ-link Sulfo-NHS-LC-Biotin reagent (Pierce) as directed by the manufacturer protocol.
Protein concentrations were determined using a Sigma QuantiPro BCA Assay Kit (Sigma-Aldrich, St Louis, MO, USA) as directed by the manufacturer's protocol and using bovine serum albumin as a standard.
The transwell invasion assay was performed with Biocoat™ Matrigel™ Invasion Chambers with 8-μm pores (BD Biosciences) as directed by the vendor's protocol.
Real-time PCR was carried out with the reverse-transcribed cDNA (2 μL) using the SYBR® Green PCR Kit (Qiagen, Hilden, Germany) in Light Cycler 2.0 (Roche, Basel, Switzerland) as directed by the manufacturer's protocol.
Reverse transcription of 10 ng of total RNA with primers corresponding to each miRNA and to U48 small nuclear RNA (RNU48) as endogenous control was performed as directed by the protocol of the two-step TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA) that incorporates a target-specific stem-loop reverse transcription primer to provide specificity for the mature miRNA target.
The sequences were as follows: (RNA -UGUGGUAGUUGGAGCUGGUGGCGUA and UACGCCACCAGCUCCAACUACCACA. LN ρ0-8, seeded at 30% confluence in a 25 cm cultuRNA -UGUGGUAGUUGGAGCUGGUGGCGUAfected with 100 ng of K-RandsiRNA or random siRNA (no. 12935-200 (Low GC), Invitrogen), UACGCCACCAGCUCCAACUACCACAmine 2000 (Invitrogen) as directed by the manufacturer's protocol.
