Isolation of single cells from the mouse heart and mediastinal lymph nodes

Mice were intracardially perfused with 50 mL of ice-cold HBSS with heparin to exclude blood cells. For heart digestion, we used a previously described protocol [24]. Briefly, the LV was dissected, minced with fine scissors, and enzymatically digested with a cocktail of type II collagenase (Worthington Laboratories, Worthington, USA) and collagenase/dispase (Roche Diagnostics, Risch-Rotkreuz, Switzerland) solution at 37 °C with gentle agitation. The cells were then passed through a 40 μm nylon mesh (BD Falcon, Franklin Lakes, USA), centrifuged (10 min, 500 g, 4 °C), resuspended in red cell lysis buffer (eBioscience, Santa Clara, USA) and incubated for 10 min. Next, the cell suspension was reconstituted with staining buffer (dPBS with no Ca²⁺ or Mg²⁺, 2% FBS). Mediastinal lymph nodes (MLNs) were isolated, homogenized, and suspended in PBS, and then passed through a 40-μm nylon mesh to remove connective tissue. The cells from digested hearts and MLNs were counted on a Countstar automated cell counter (Rui Yu Biotech, Shanghai, China).