Facscelesta cell analyser

Neonatal CD1 mice at age p1 randomly received AAV9-MCS or AAV9-miR106b~25. After 10 days, all mice we administered a single EdU injection and 2 days later cardiomyocytes were isolated and fixed using 4% PFA. Next, cells were permeabilized using 0.1% Triton-X and incubated for 2 h at room temperature with mouse monoclonal antibody [EA-53] to sarcomeric alpha-actinin (1:100, Abcam ab9465), followed by an incubation of 1 h with goat anti-mouse IgG secondary antibody Alexa Fluor-488 conjugated (1:100, ThermoFisher A-11001). Next, cells were further processed using the Click-IT EdU 647 Imaging kit to reveal EdU incorporation, according to the manufacturer’s instructions, and stained with Hoechst 33342 (Life Technologies). Acquisition and analysis was performed on a BD FACSCelesta cell analyser. Analysis was performed using FACSDiva Version 6.1.3.

PANC-1 cells were harvested from the culture dish by removing the culture media and washing once with PBS before detaching by incubating with Trypsin-EDTA (Sigma-Aldrich, Merch UK). Once ≥90% of cells were detached, cells were washed once with PBS and stained with 5uM BODIPY 493/503 (#D3922, Invitrogen, USA) in PBS and incubated in the dark for 15 minutes at 37 °C with 5% CO₂. After incubation, cells were washed and resuspended with PBS with 5% FBS before being analysed using a BD FACSCelesta™ Cell Analyser. Data analysis was performed using FlowJo (BD Life Sciences, UK). The experiments were biological replicates of n = 3.