Fluoview analysis software

Confocal Ca²⁺ imaging experiments were performed with an Olympus Fluoview laser-scanning confocal microscope (Olympus Life Science, Center Valley, PA, USA) as previously described [13 (link)]. Fluo 4-AM (dissolved in 20% F-127 pluronic in dimethyl sulfoxide (DMSO), final concentration 15 μM, Molecular Probes, Eugene, OR, USA) was added to hiPSC-CMs and incubated for 20 min at RT. Fluo-4 loaded hiPSC-CMs were placed in a perfusion chamber and excited at 488 nm using an argon laser, and fluorescence emission was detected via a 520-nm band-pass filter and photomultiplier tube. Confocal images were acquired with Fluoview acquisition software program and spontaneous activity was recorded on personal computer for later analysis. Images acquired with Fluoview acquisition software were analyzed with ImageJ and Fluoview analysis software (Olympus Life Science, Center Valley, PA, USA).

B6 and CD80^−/− mice were treated with LPS or left untreated. Twenty-four hours later, kidneys were snap frozen in liquid nitrogen with optimum cutting temperature (OCT) embedding medium (Cryomatrix, Thermo Scientific). Cryosections 3-µm thick were fixed in ice-cold methanol. After blocking with 1% BSA in PBS, primary antibodies against CD80 (R&D systems, catalogue no. AF740, 1:40) and synaptopodin (Progen, Germany, catalogue no. 61094, 1:40) were added. Sections were washed with PBS-Tween-20, followed by staining with appropriate secondary antibodies (anti-goat-Ig coupled to AlexaFluor488 for CD80 detection and anti-mouse-Ig AlexaFluor568 for synaptopodin detection, Life Technologies; catalogue nos. A11078 and A11004, respectively, at 1:500 dilution). Sections were stained with DAPI (Sigma) and mounted with 65% glycerol (Sigma). Images were taken using an Olympus FV1000 confocal laser scanning microscope. Images were acquired using a 60× objective (NA 1.42) with 2× optical zoom. AlexaFluor488 and AlexaFluor568 were excited using green and red LED lasers, respectively. Serial z-stack images of 0.4-µm sections were acquired. The images were processed using Olympus fluoview analysis software.

GraphPad Prism software version 5 was used for statistical analysis and graph drawing. Where stated, graphs were created using the Olympus FluoView analysis software. De Novo software version FCS Express 6 was used for flow cytometry analysis. One way analysis of variance (ANOVA), and student unpaired t-test were used to test statistical difference between two groups. Bonferroni's test was chosen as a post hoc test. Data were represented as mean ± standard deviation (mean ± SD) when biological independent replications was three to six (n =3-6) and significant difference value was less than 0.05 (P < 0.05).