Protocols for Isolating Mouse Cardiomyocytes

Mouse hearts were isolated for single cell preparation as previously described^{9 (link)} with atria and valves removed. Isolated mouse hearts were digested using one of three protocols (designated Protocol 1, 2 and 3). For Protocol 1, each mouse heart was divided in approximately 20 pieces, placed in 10 ml of Protocol 1 digestion buffer [2 mg/ml collagenase type II in 1× HBSS (Worthington Biochemical Corporation)] in gentleMacs C-tubes (Miltenyi Biotec), dissociated using Heart 1 program of a gentleMacs Dissociator, and incubated at 37° C for 40 min with gentle agitation. Following incubation, tissue was further dissociated using Heart 2 program before placing on ice. For Protocol 2, isolated hearts were finely minced using forceps to ~2 mm pieces and placed in 3 ml of Protocol 2 digestion buffer [2 mg/ml collagenase type IV (Worthington Biochemical Corporation) and 1.2 U/ml dispase II (Sigma-Aldrich or Thermofisher Scientific) in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.9 mM CaCl₂]. Tissue was incubated at 37° C for 15 min with gentle rocking. Following incubation, tissue digestion buffer with tissue clusters was triturated by pipetting 12 times using a 10 ml serological pipette. Dishes were again incubated at 37° C and triturated twice more (45 min of total digestion time). The final trituration was conducted by pipetting 30 times with a p1000 pipette. For Protocol 3, isolated mouse hearts were finely minced using forceps, placed in 10 ml Protocol 3 digestion buffer [125 U/ml collagenase type XI (Sigma-Aldrich), 60 U/ml hyaluronidase type I-s (Sigma-Aldrich), and 60 U/ml DNase 1 (Sigma-Aldrich) in DPBS supplemented with 0.9 mM CaCl₂ and 20 mM HEPES] incubated at 37° C for 1 hour with gentle agitation, triturated 20 times using a 10 ml serological pipette, and placed on ice. All cell suspensions were filtered using a 40 µm cell strainer. Filtered suspensions were placed into 50 ml tubes with 40 ml of DPBS and centrifuged at 200 g for 20 min with centrifuge brakes deactivated to remove small tissue debris. Cell pellets were resuspended in 250 µl 2% FBS/HBSS solution before staining with various antibodies and reagents for flow cytometry or FACS.