Te300 fluorescent microscope

hPASMC were seeded on 24-well plates containing gels with stiffness of 0.4 and 25.6 kPa and treated with iloprost (10 μmol/l) or vehicle (DMSO). After 48 hours, cells were fixed with 4% formalin, blocked with 5% goat serum, and stained with mouse monoclonal anti-Pro-Collagen I (clone PCIDG10, catalog MAB1913, Millipore) and mouse monoclonal anti-Fibronectin (clone IST-9, Abcam) antibodies. After washing, cells were incubated with Alexa Fluor 546 goat anti–mouse IgG1 (Invitrogen). F-actin and nuclei were stained with Alexa Fluor 488–phalloidin and Hoechst 33342 (Invitrogen), respectively. Fluorescent images were obtained with a Nikon TE300 fluorescent microscope. The final image contrast was adjusted with MetaMorph 6.1 (Universal Imaging) by setting the background to the fluorescence intensity level of the samples in the corresponding secondary antibody–only control images. Procollagen I staining was quantified with MetaMorph 6.1 by counting the procollagen I staining–positive cells and normalizing to the total cell number in each image. Fibronectin staining was quantified with MetaMorph 6.1 by measuring the average cellular fibronectin fluorescence intensity (normalized to stained areas) and then normalized to the corresponding total cell number.

HEK293 cells were seeded in 24 well plates. Upon reaching 90% confluency cells were co-transfected with 400 ng of GFP and 400 ng of effector plasmid. The transfection cocktails were prepared as described above. After 48 h post-transfection cells were viewed by a Nikon TE300 fluorescent microscope. Next, cells were resuspended in 0.5% BSA and assayed by flow cytometry following the commercial protocol using the Millipore Guava EasyCyte plus flow cytometer. The mean fluorescent intensity (MFI) was calculated using FlowJo (TreeStar) software.