We describe a protocol for microdissection and subsequent isolation of cardiac resident microphages by fluorescence-activated cell sorting specifically from sinus and AV node in the mouse. With the precise microdissection of the sinus and the V node, followed by the combination of magnetic and fluorescence-activated cell sorting, we are able to specifically isolate resident macrophages from the sinus and the V node at high purity. Identification and isolation of cardiac resident macrophages from distinct regions in the heart allows to further study their phenotype and their region-specific impact on cardiac electrophysiology.
After isolating the heart, perform the microdissection procedures in a dissection dish with ice cold one-time PBS under a dissecting microscope. Use cardiac anatomical landmarks such as aorta, pulmonary artery, coronary sinus, and left or right ventricle to determine the left-right and anterior-posterior of the heart. After the orientation is determined, position the heart with the front at the bottom of the dish to expose the posterior large veins.
Cut along the groove between the ventricles and the atria to remove the majority of the ventricles. Keep the atrial part. Fix the heart to the dissection dish by inserting pins through the remaining part of the LV free wall and the LAA.
Cut the remaining RV free wall upwards through the tricuspid valve and superior vena cava. Flip the RA and RV away and pin the RAA to expose the RA cavity intercaval region and atrial septum. For the microdissection of SAN, cut the heart along the interatrial septum parallel to the crista terminalis to separate the intercaval region to obtain the SAN sample.
Put the sample in an empty 1.5 milliliter microcentrifuge tube on ice. For microdissection of AVN, pin the remaining parts of the heart through the tissue adjacent to the interatrial septum and interventricular septum to face the right atrial side of the interatrial septum up. Look at the right atrium on the endocardial surface for the triangle of Koch which will be bordered anteriorly by the hinge line of the septal leaflet of the tricuspid valve and posteriorly by the tendon of Todaro.
The orifice of the coronary sinus is observed at the base. Mince the SAN and AVN tissue with scalpels. Then add 500 microliters of freshly prepared digestion buffer to each sample and wash all minced tissue from the wall of the microcentrifuge tube.
Gently pipette to help digest the sample and homogenize the tissue on a vortex machine. After digestion, transfer the tissue suspension to a fresh 15 milliliter centrifuge tube by passing it through a 40 micrometer cell strainer. Rinse the cell strainer with an additional five milliliters of cell sorting buffer to stop the digestion.
Remove the supernatant. After resuspending the cell pellet with 90 microliters of cell sorting buffer, add 10 microliters of CD45 microbeads per 10 million total cells to the cell suspension in a 15 milliliter centrifuge tube. Mix the samples well.
During the time for incubating the cell suspension with microbeads and antibody mixture, prepare the magnetic separation set by attaching the magnetic column to a suitable magnetic separator and then placing a collection tube under the magnetic column. Prepare magnetic columns by rinsing with 500 microliters of cell sorting buffer added at the top of the column and allowing the buffer to pass through. Apply the cell suspension immediately onto the column, avoiding air bubbles, while the cell sorting buffer is passing through.
Wash the column three times with 500 microliters of cell sorting buffer. Add one milliliter of cell sorting buffer onto the column. Remove the column from the magnetic separator and place it on a new collection tube.
Immediately flush the column by firmly applying the plunger supplied with the column to obtain the flow-through containing the magnetically labeled cells. After applying the unstained sample and compensation tubes, adjust the voltages of each channel to align both the positive and negative peaks to the proper position of the axis. Set the gating strategy as described in the text manuscript using DAPI as a viability marker.
Check the flow cytometry charts to confirm that the cell population of interest is properly shown on the charts. Collect the sorted cells into medium or buffer-based on the need of subsequent experiments. To confirm a correct dissection, identification of the SAN and AVN was done using immunofluorescent and Masson's trichrome staining.
Immunofluorescent staining of HCN4 positive conduction system cells in the SAN and AVN, as well as Masson's trichrome staining of SAN and AVN is shown here. The gating strategy for cell sorting of resident cardiac macrophages helped to identify mononuclear cells by exclusion of doublets in the forward scatter area of the y-axis versus that of the x-axis. Dead cells were excluded by DAPI.
Live cells were gated for CD45 positive leukocytes, then for CD11b positive myeloid cells. Cardiac macrophages were identified by the expression of both F4/80 and CD64, then stratified by lymphocyte antigen six expression. Freshly sorted cells were observed under brightfield microscopy.
The cells were positive for CD45 when observed under the fluorophore PE channel. When observed under fluorophore APC size seven channel, the same view of the sorted cells showed CD11b positive cells. The fluorophore PE size seven channel was used to identify the F4/80 positive phenotype.
And the triple positive cells were identified as cardiac resident macrophages. Isolated cardiac macrophages cultured in medium for up to six days are indicated with white arrows, while the black arrows indicate dead cells. Sorted macrophages can be used for further functional studies such as patch clamp experiments or expression studies such as RNA sequencing or proteomic studies.
