Coulter epics xl mcltm

Induced pluripotent stem cells were trypsinized, washed in cold PBS, and fixed for 20 minutes at RT in 3.7% formaldehyde. Cells were permeabilized with 0.4% Triton X-100 in PBS and subsequently incubated with antibodies as indicated for 2 hours at 37°C in 0.4% Triton X-100 in PBS. Stainings were performed with antibodies against cardiac Troponin I (BD Pharmingen, San Diego, US), αMHC (BD Pharmingen, San Diego, US), and α-actinin (Sigma Aldrich, St. Louis, US). A FITC conjugated IgG antibody served as a control. After washing, cells were analyzed on a Coulter Epics XL-MCLTM flow cytometer (Beckman Coulter, Krefeld, Germany).

Cell viability and cell cycle progression were examined by flow cytometry to measure the amount of DNA in nuclei stained with propidium iodide (PI; Sigma-Aldrich, Milan, Italy), with the exclusion of necrotic cells by forward light scatter (FSC) [66 (link)]. Briefly, cells were harvested by centrifugation and gently resuspended in 1.5 mL hypotonic PI solution (50 µg/mL in 0.1% sodium citrate plus 0.1% Triton X-100). Tubes were kept in the dark at 4 °C for 30 min. PI fluorescence of individual nuclei was measured by flow cytometry using a Coulter Epics XL-MCLTM flow cytometer (Beckman Coulter, Cassina De’ Pecchi, Milan, Italy) and analysed using FlowJo_V10 software (BD Biosciences, Milan, Italy).