Aria 2 sorp cell sorter

To isolate the cell population with a high ALDH enzymatic activity, ALDEFLUOR assay kit (STEMCELL Technologies Inc.) was used according to the manufacturer’s instructions. After trypsinization, cells were suspended in ALDEFLUOR assay buffer containing ALDH enzyme substrate BODIPY-aminoacetaldehyde (BAAA), and incubated at 37°C for about 40 minutes. Cells were stained using the identical conditions with the specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), to serve as a negative control. Flow cytometry sorting was conducted using a BD Bioscience Aria II SORP cell sorter.

EM analysis after FACS (FACS-EM) was performed as follows. Isolated CMs in suspension were fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were next filtered by passing through a 100 µm cell strainer, pelleted by centrifugation at 20 × g for 5 min at room temperature, and resuspended in ~1 ml perfusion buffer. FACS was performed using a BD Aria II SORP cell sorter with a 100 µm nozzle. After FACS, the cells were fixed again in a mixture of 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, overnight at 4 °C. The cell pellets were next processed through a routine transmission EM (TEM) protocol at Harvard Medical School EM core. Images were taken using a JEOL 1200EX-80 kV EM. Because of the cell size and stiffness, fixed adult CMs easily clogged the FACS machine. Currently, FACS-EM only works for CMs from P30 and younger mice.

To establish cell lines stably expressing fluorescent markers, we subcloned EGFP (enhanced green fluorescent protein) cDNA and mCherry cDNA into the pcDNA3.0/Neo vector. pcDNA.EGFP/Neo and pcDNA.mCherry/Neo were transfected into SKBR3 and SK/Her^R cells, respectively, using Lipofectamine LTX/PLUS (Invitrogen by Life Technologies) according to the manufacturer's instructions. Stably transfected populations (SKBR3.EGFP and SK/Her^R.mCherry) were selected and maintained in 1 mg/mL G418. To make the pcDNA.tDp/mCherry/Neo vector, t-Darpp cDNA with a 2A peptide sequence [38 (link)] attached to the C-terminus was subcloned into the pcDNA.mCherry/Neo vector upstream of and in-frame with the mCherry sequence. The plasmid was then transfected into SKBR3 cells and the stably transfected population (SK.tDp2A.mCherry) was selected and maintained in 1 mg/mL G418. Each transfected cell population was fluorescently sorted via flow cytometry (using the Aria II SORP cell sorter from Becton Dickinson, Franklin Lakes, New Jersey) to isolate the cell populations with the highest fluorescent protein expression. Sorted cells were characterized as described in the Results and Discussion section and then used in all subsequent co-culture experiments.