Spectra max m2 microplate reader

Exosomes from control or SYTO-RNA-labeled HSC or that contained Cy3-miR-199a-5p were labeled for 1 hr with 4 μM of the fluorescent lipophilic membrane dyes PKH26 or PKH67, according the manufacturer’s specifications (Sigma-Aldrich). Exosomes (0- 4 μg/ml) or free Cy3-labeled miR-199a-5p (1 μM) were added for up to 48 hrs to primary mouse HSC or hepatocytes which were then washed in PBS and imaged using a confocal microscope (Zeiss, Obercochen, Germany) or lysed in lysis buffer (Boston Bioproducts, Ashland, MA) and measured at 590/540 nm using a Spectra Max® M2 microplate reader (VWR, Atlanta, GA) to assess levels of PKH26 flourescence. Prior to exosome addition in some experiments, HSC were stained with PKH67 (Sigma-Aldrich) and hepatocytes were stained with far red (Sigma-Aldrich). In some binding experiments, HSC were pre-treated or co-incubated with 0-100 μg/ml RGD or RGE tripeptides (American Peptide, Sunnyvale CA), 0-100 μM EDTA (Sigma-Aldrich), 0-10 μM sodium chlorate (Sigma-Aldrich), 0-10 μM sodium sulfate (Sigma-Aldrich), 0-10 μg/ml rabbit anti-mouse integrin αvβ3 IgG (Bioss Inc, Woburn, MA), or 0-20 μg/ml rat anti-mouse integrin α5β1 IgG (Millipore, Temecula, CA) or 0-10 μg/ml rat anti-mouse integrin αM, (CD11b; Novus Biologicals Littleton CO). For antibody studies, non-immune IgG was used as a negative control.