Alexa 647 secondary antibodies

Rat neonatal CMs were fixed in 4% paraformaldehyde. Anti-cardiac troponin I (CTNI) 1:500 (ab56357, Abcam) and anti-PKM2 1:100 (D78A4, Cell Signaling) were used. After washing with PBS, samples were stained with Alexa-568, Alexa-488 or Alexa-647 secondary antibodies 1:500 (Life Technologies), followed by 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) 1:2000 (Merck) staining to visualize DNA.

Approximately 250 cells per well for each cell line were seeded in each well of 1,536-well microtiter plates (PerkinElmer) and incubated overnight. Combinatorial drug library was prepared as 300X stocks in 384 well plate and 20nL pin-transferred to 1,536-well plates. Plates were incubated for 72 hours and fixed in 4% formaldehyde, washed in PBS containing 0.1% Triton, and incubated overnight with antibodies (1:500) to cleaved PARP (above). The plates were then washed twice, incubated with Alexa 647 secondary antibodies (1:1000, Life Technologies) and DAPI 2–16 hours, and washed with PBST. Plates were then imaged using the Cellworx high-throughput microscope (Applied Precision Inc.) and counting nuclei and cells with cPARP staining with the Multi-Wavelength Cell Scoring module of the MetaExpress image analysis software (Molecular Devices). Z’ values were between 0.8–0.9 for most cell lines.