Anti α actinin

iPSCs were co-cultured with murine visceral endoderm-like (END-2) cells (Humbrecht Institute, Utrecht, The Netherlands) to differentiate them into spontaneously beating CMs. The beating areas of the cell colonies were mechanically excised and treated with collagenase A (Roche Diagnostics).[28 (link)] Single CMs were immunostained with anti-cardiac-troponin-T (1:1500, Abcam, Cambridge, MA, USA), anti-α-actinin (1:1500, Sigma) and anti-connexin-43 (1:1000, Sigma).

CMs were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde dissolved in PBS for 20 min. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, washed in PBS + 0.1% Tween 20 (PBST), and blocked with 5% normal goat serum (NGS, Thermo Fisher Scientific) in PBST. The cells were stained with the following primary antibodies: anti-cTnT (Thermo Fisher Scientific), anti-α-actinin (Sigma-Aldrich), anti-MLC2v (Proteintech, Rosemont, IL, USA), anti-MLC2a (Synaptic Systems, Goettingen, Germany), and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA) antibodies at 4 °C overnight in 2% NGS in PBST. The cells were washed twice in PBST and incubated for 1 h with the following secondary antibodies: Alexa Fluor 488 anti-mouse IgG1, Alexa Fluor 594 goat anti-mouse IgG2b, and Alexa Fluor 594 goat anti-rabbit IgG (all from Molecular Probes, Eugene, OR, USA). The nuclei were stained with DAPI, and the stained cells were mounted using a fluorescent mounting solution (DAKO, Carpinteria, CA, USA). Immunofluorescence images were acquired using a fluorescence microscope (Olympus-Europa GmbH, Hamburg, Germany) and a confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Cultures were fixed in 4% paraformaldehyde (PFA, Gibco^®) and permeabilized in 0.5% Triton X-100 in PBS. Protein blocking was completed using 5% bovine serum albumin (BSA, Sigma©) in 0.1% Triton X-100. Primary antibodies anti-desmin (1:50, Sigma©), anti-MyoD (1:100, BD Biosciences), anti-α-actinin (1:500, Sigma©) and anti-MyHC (1:1, DSHB) were incubated overnight at 4 °C. Secondary antibody Cy3-conjugated sheep anti-mouse (1:1000, Sigma©) was incubated for 1 h at room temperature in the dark. Cell nuclei were stained with DAPI (1:100, Sigma©). Images were acquired with Leica DM6000 B microscope and LAS AF software both from Leica Microsystems Inc.

To prepare cells for flow-based analysis, cells harvested using trypsin was fixed and permeabilized by resuspension in 250μL BD Cytofix/Cytoperm™ solution (BD Biosciences #554722) for 20 minutes at 4°C. Cells were washed twice with 1mL BD Perm/Wash™ buffer (BD Biosciences #554723) and incubated with anti-cardiac troponin T (anti-cTnT) (ThermoFisher Scientific #MA5-12960) and anti-α-actinin (Sigma-Aldrich #A7811) at dilutions of 1:100, 1:800 respectively for 30 minutes at 4°C. Following 2 washes with BD Perm/Wash buffer, cells were labeled with DyLight633-conjugated goat anti-mouse IgG (ThermoFisher Scientific #35513) at 1:250 dilution for 30 minutes. Following 2 more washes, cells were resuspended in 200μL of BD Perm/Wash buffer, and flow data acquired using the BD Accuri™ C6 flow cytometer set for 30,000 events. Post-acquisition analysis was conducted using FlowJo v10.2 (BD Biosciences).
To calculate differentiation efficiency, the following scheme was applied. Flow data was gated into 4 quadrants, with Q1 and Q4 designated as GFP_-ve cells, while Q2 and Q3 are GFP_+ve cells. In addition, cells in Q1 and Q2 are designated as cardiomyocyte populations. Thus, the % differentiation efficiency of GFP_+ve cells is calculated as Q2/(Q2+Q3)*100; while general differentiation efficiency (label independent) is calculated as (Q1+Q2)/(Q1+ Q2+Q3+Q4)*100.