Protocol for Isolation of Cardiac Interstitial Cells from Adult Murine Hearts for Unbiased Single Cell Profiling

Figure 1. Workflow Graphic summary of the main steps to obtain live nucleated single interstitial cells from adult murine hearts.

Figure 2. Images of Critical Steps (A) Images of the set up before the experiment, which include preparing the buffers and turning on the AutoMACS; preparing the ice buckets, the collection tubes, the dissection stage; refrigerating the centrifuges at 4°C and warming up the water bath at 37°C. (B) Images showing the dissection of the ventricles, enzymatic digestion in Erlenmeyer flask on a magnetic stirrer and collection of the cell suspension in a collection tube (CT) on ice. (C) Images of the Red blood cell (RBC) lysis step and the pellet suspension in dead cell removal beads.

Figure 3. Images of Critical Steps (A) Image of the AutoMACS. The orange arrow indicates the intake port and the white arrow indicates the output or negative selection port (“neg”). (B) Schematic representation of the autoMACS main screen. (C) Workflow indicating the steps to perform on the AutoMACS per each sample. (D) The eluted cell suspension is divided in two 5 mL FACS tube and centrifuged. (E) Each pellet is resuspended in in 500 μL of FACS buffer with DRAQ5. PI is added after 5 min, before sorting. (F) Full gating strategy for the selection of single live (PI-) nucleated (DRAQ5+) cells.

Figure 4. Additional Combination of Dyes Tested for the Selection of Live Nucleated Cells (A) FACS plots showing cells labelled with DAPI (180nM) and 3 serial dilutions of DRAQ5 (0.1, 1, 10 μM). (B) FACS plots showing the gating strategy for cells co-labeled with PI, CalceinV450, and DRAQ5. Cells in Q1: Calcein +PI- (live metabolically active cells) and Q4: Calcein-PI- were further subclustered based on the DRAQ5 expression (right panels, top and bottom, respectively). Percentages of populations are reported on each plot and presented as average ± SEM of 3 replicates.