To begin, obtain electro operated mouse hearts extracted from wild type CD1 mice, 12 to 12.5 days after crossing. Transfer a heart into a tube containing one milliliter of ice cold digestion medium. With a syringe, apply gentle pressure on the heart a few times to chop it.
Incubate the chopped tissue on a hot plate at 37 degrees Celsius for 45 minutes at 600 rotations per minute. Then filter the digestion mixture through a 70 micrometer cell strainer into a new tube. Next, filter the pass through volume again with a 40 micrometer cell strainer into a new 50 milliliter tube.
Pipette 500 microliters of the FBS into the filtrate. Make up the volume up to 30 milliliters with cold DMEM. Centrifuge the suspension at 240G for 10 minutes at room temperature.
Discard the supernatant. Then place the tube upside down on a paper towel to dry completely. For cell sorting, resuspend the cells in 300 microliters of sorting buffer.
Finally, add 0.3 microliters of DAPI and perform cell sorting. The hearts were beating and appeared to be in good condition 24 hours post electroporation. The myocardium and epicardium did not show any signs of apoptosis.
Analysis of the fluorescence activity showed a mosaic GFP signal in almost all four chambers of the heart.
