Protocol for quantifying murine cardiomyocyte cell division by single-cell suspension

Figures

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  Figure 1. Schematic of the Langendorff digestion process (1) Set up your dissection workstation. (2) Euthanize animal, pin down in supine position, and open thoracic cavity. (3) Dissect out the heart and rinse in Petri dish of KB solution. (4) Transfer heart to second Petri dish of KB solution und the dissection scope. Scope workstation should have a pre-mounted cannula with a loose knot slipped over it. (5) Cannulate the aorta and tie a knot around the aorta-cannula. After knot is secured and pulled tight, remove atria. (6) Hang from Langendorff apparatus with calcium-free Tyrodes preloaded. Digest with designated volume of 1 mg/mL collagenase dissolved in calcium-free Tyrodes. (7) Cut heart off the cannula into a dry Petri dish, careful to take the majority of the ventricles leaving behind the outflow tract. (8) Dice up heart with scissors. (9) Add 1 mL of KB solution to diced ventricular tissue and triturate with a wide bore pipette. (10) Transfer digested tissue in KB to a 2 mL microcentrifuge tube and add an equal volume of 8% PFA. Fix for 10 min. (11) Spin cells down at 300 g for 2 min. Remove supernatant and resuspend pellet in PBS. Figure made in BioRender.

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  Figure 2. Example data collected for nucleation under the microscope

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  Figure 3. Example data exported to excel by NIS Elements software Columns A–D are exported directly from NIS elements. Column E indicates the nucleation status which you manually add by returning to the picture, finding the ObjID and recording if the object comes from a mono-, bi-, tri-, or tetra-nucleated cardiomyocytes (Step 54a). Column F is manually entered EdU status (Step 54b). Column G is the estimated ploidy value based on a normalization value for that picture (Step 56–57). Column H is the binned ploidy value (Step 58). Column I is the calculated normalization value for a single picture based on tri- and tetra-nucleated diploid nuclei (Step 56).

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  Figure 4. Example data of EdU-positive cardiomyocytes for steps 62 & 63 of Major Step: Ploidy analysis

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  Figure 5. Example data for 64 & 65 of Major Step: Ploidy analysis The EdU-positive 1 × 2N cells must have completed cell division. Thus, in this example, 4% of those cells that underwent DNA synthesis in the labeling period completed cell division.

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  Figure 6. Schematic of total CM number by hemacytometer (1) Take three small aliquots of ventricular prep from Major Step: Langendorff Procedure of this protocol. Dilute 1:5 in PBS. Be sure to remix the cell preparation between each aliquot. (2) Collect 10 μl of your dilution and (3) load it into the chamber of a standard hemacytometer. (4–6) Under an upright phase microscope quantify the number of CMS in the 4 × 4 frames. Red arrowheads point to CMs that should be excluded as they are touching an outer boundary of the grid. Green arrowheads point to CMs that should be included as they are touching the inner boundary of the grid. Repeat this 3 times for each of the 3 aliquots (i.e., 9 data points to average for one single sample). Figure made in BioRender.