This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.
This method can help answer key questions about the composition of immune cell subsets within the heart both during steady and disease states. The main advantage of this technique is that it can be used for accurate immunophenotyping of cardiac immune cell populations, including very rare subsets. This protocol's relatively simple.
However, tail vein injections can be difficult, so it's important to practice this technique until you're comfortable before using experimental animals. To label the circulating leukocytes, load 200 microliters of freshly-prepared mouse fluorophore-conjugated anti-CD45 antibody in PBS into a 28.5-gauge insulin syringe, and prewarm eight 12-week old mice on a circulating water blanket set to 35 to 41 degrees Celsius for five to ten minutes. When the tail vein has dilated, restrain the first animal in the sternal position in an appropriate restraining device.
If the tail veins are not sufficiently dilated, dip the tail in 36 to 40-degree Celsius water for one to two minutes. When the tail veins are ready swab the injection area with 70%ethanol-soaked gauze and immobilize the base of the tail with the index and middle fingers and the mid-distal region of the tail with the thumb and ring finger. Holding the needle bevel-side up and parallel to the vein, insert the needle into a dilated tail vein and intravenously inject the entire volume of antibody.
Then allow the antibody to circulate for five minutes before humane euthanization. To harvest the heart, spray the mouse with 70%ethanol and lift the skin of the abdomen to open the abdomen along the ventral midline from the hips up to the sternum. Open the peritoneum and move the liver to expose the diaphragm.
Cut the diaphragm and the ribs on both sides of the midline, taking care to avoid puncturing the lungs and heart. Peel back the ribs to expose the heart before incising the left and right atria. Gently grasping the base of the heart with forceps, insert a 21-gauge needle attached to a 60-milliliter syringe into the left ventricle near the apex and perfuse the heart with 15 to 20 milliliters of cold PBS.
Proper perfusion of the heart is essential to avoid intravascular contaminants. Therefore take care to dissect and perfuse the heart quickly after euthanasia to avoid contamination to due to coagulated blood. After perfusing the right ventricle as just demonstrated, white lungs and a pale liver should be observed.
Holding the base of the heart with the forceps gently pull the heart up and cut the major pulmonary arteries and veins, aorta and pericardial sac to detach the cardiac tissue. Gently holding the heart between the thumb and index fingers carefully clean heart with a clean lint-free paper towel and use the forceps to remove the atria and any remnants of any major arteries and veins or other contaminants. Place the cleaned heart in a five-centimeter plastic dish and add 50 microliters of cold PBS.
Using curved dissecting scissors mince the heart until there are no visible pieces and the tissue has a smooth thick consistency. Use curved forceps to transfer the tissue into two-milliliter microcentrifuge tubes containing 800 microliters of cold DMEM. Supplement with collagenase, DNase, and hyaluronidase and incubate for 45 to 60 minutes at 37 degrees Celsius and 50 rpm.
At the end of the digestion vortex samples for 20 seconds and immediately place them on ice. The sample should always be kept at four degrees Celsius unless otherwise specified as some surface antigens can be internalized if left at room temperature. Then pre-wet 140-microliter cell strainer in a 50-milliliter conical tube per sample with one milliliter of cold HBB.
Using a one-milliliter pipette, pipette the samples until the tissue suspensions are homogenous and do not clog the pipette tip and add the homogenized samples to the filters. Slowly wash the samples through the filters with 12 to 14 milliliters of cold HBB per sample, and transfer the filtered samples to a labeled 15-milliliter conical tube on ice. A good digestion is demonstrated by the absence of heart remains in the filter.
Collect the samples by centrifugation and remove the supernatant. Add one milliliter of ammonium chloride potassium lysis buffer to each sample. Resuspend the samples with brief vortexing, followed by a five-minute incubation at room temperature.
Then stop the hemolysis with five to eight milliliters of cold HBSS. Collect the samples by centrifugation and resuspend the pellets in one milliliter of fluorescence-activated cell sorting or FACS buffer and transfer to individual 1.5-milliliter microcentrifuge tubes for another centrifugation. To analyze the samples by flow cytometry resuspend the pellets in 50 microliters of anti-CD16/CD32 antibody and a monocyte blocker to reduce the nonspecific antibody binding for a 15-minute incubation at room temperature.
Without washing, add 50 microliters of antibody master mix to each sample with mixing for a 30-minute incubation at four degrees Celsius protected from light. At the end of the incubation, wash the samples with 600 microliters of FACS buffer per tube and resuspend the pellets in 300 microliters of fresh FACS buffer or 1%paraformaldehyde. Then analyze the cardiac leukocytes by flow cytometry according to standard flow analysis protocols.
Analysis of the cardiac single cell suspension by flow cytometry requires pre-gating with CD45 to differentiate immune cells from nonimmune cells of the heart, followed by single cell and small size exclusion gating. Macrophages, identified by their CD64 expression, are the major immune cell population of the heart, representing 60 to 70%of all immune cells. Conventional cardiac dendritic cells are CD64-negative cells that express high levels of MHC class II and CD11c.
These cells can be further divided based on their expression of either CD103 or CD11b and represent a much smaller cardiac subpopulation compared to macrophages. The use of flow cytometry of a single cell suspension of myocardium allows the study of small leukocyte subpopulations within the heart. For example, using flow cytometry analysis to compare the immune cells of the myocardium of wild type versus Batf3-deficient mice, the lack of CD103-positive dendritic cells can be confirmed within the knockout animals.
In addition, this analysis revealed a lack of significant alteration of the composition of other cardiac populations in the Batf3-deficient animals, demonstrating the degree of sensitivity that can be achieved using this combined method of enzymatic and mechanical digestion to achieve a single cell suspension. While attempting this procedure, it's important to always take into account the viability of your cell isolate. Therefore we recommend optimizing the time and enzyme concentration of the digestion according to your experimental conditions.
Following this procedure, other methods, such as single cell RNA sequencing, functional assays, and magnetic bead isolation can be performed to answer additional questions about the functions of the isolated cells.
