Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Our research focuses on the role of immunometabolism during myocardial infarction. We wanna understand how changes in immune cell metabolism regulate their phenotype during cardiac injury and inflammation.

We combine the in vivo genetic approaches with precision cell extraction, cell phenotyping, metabolic phenotyping, transcriptomics, and metabolomics to better understand the impact of macrophage metabolism on cardiac remodeling.

This protocol allows us to understand how macrophage metabolism changes after MI, under different disease conditions such as obesity or diabetes, and how different therapies affect macrophage metabolism.

This technique allows rapid analysis of freshly extracted live macrophages, thus avoiding long-term culturing. It also allows the simultaneous assessment of other phenotypic changes in the same cells.

This protocol will allow us and others to further understand macrophage metabolism across different inflammatory or autoimmune states. It could also be modified to study other immune subtypes.

[Narrator] To begin, obtain the leg bones from the experimental mouse, and carefully cut off both ends of the bone. Using a 10-milliliter syringe with a 27-gauge needle, flush the bone marrow into a 15-milliliter conical tube containing PEB buffer. Centrifuge the suspension at 300 G for 10 minutes at four degrees Celsius. Generate a single cell suspension and lyse the RBCs. Then add PEB buffer, followed by Fc receptor blocking reagent and monocyte biotin antibody cocktail to five times 10 to the power of seven total cells. Incubate the mixture in the dark at four degrees Celsius for five minutes. Wash the cells with 10 milliliters of PEB buffer, and centrifuge the suspension at 300 G for 10 minutes at four degrees Celsius. Next, collect the cell pellet and resuspend it in 500 microliters of PEB buffer and 100 microliters of anti-biotin microbeads. Incubate the mixture in the dark at four degrees Celsius for 10 minutes. Now, prepare a magnetic column on a stand with one milliliter of PEB buffer. Immediately apply the cell suspension to the column, and collect the flow through containing monocytes in a 15-milliliter tube. After washing the column twice with one milliliter of PEB buffer, count the monocytes in the flow through using a hemocytometer. To prepare the monocytes for the next step, centrifuge them at 300 G for 10 minutes at four degrees Celsius. For flux analysis, resuspend the cell pellet in one milliliter of RPMI with 0.1% FBS. Keep the remaining cells in cold PEB for flow cytometry and proceed with staining. A day before the assay, remove the sensor cartridge from its packaging. Detach the green sensor plate from the wells and pipette 200 microliters of calibrant fluid into each well. Place the sensor plate back onto the cartridge plate so the sensors are submerged in the calibrant fluid. Place the cartridge in a non-carbon dioxide humidified incubator at 37 degrees Celsius overnight. Then plate the cells along with 200 microliters of media into a 96-well culture plate for at least one hour. Prepare the metabolic flux media for the assay. Use basal RPMI or DMEM without glucose, glutamine, or pyruvate, and supplement the media according to the specific test. Carefully replace the old media in the wells with the newly prepared basal media, and check the cells under a microscope to confirm their integrity. Place the cell plate in a non-carbon dioxide humidified incubator at 37 degrees Celsius. Now retrieve the hydrated sensor cartridge flux plate from the incubator, and prepare the test compounds for glycolysis or mitochondrial stress tests. Load appropriate volumes of test compounds into ports on the flux plate. Now open the software and select Mitochondrial Stress Test. Select the wells to be used, ensuring that at least one well is blank for background measurements. To run the program, load the flux plate without its lid for calibration, which takes approximately 20 minutes. After calibration, remove the bottom of the flux plate and place the cell plate in the holder. Press Run to start the assay, which will take approximately 2.5 hours. Analyze the results using metabolic flux software or other analytical tools. The bone marrow from two tibias and two femurs yielded a good amount of monocytes. Flow cytometry illustrated cardiac and splenic macrophage heterogeneity for Ly6C, while bone marrow monocytes were highly positive for Ly6C. Changes in extracellular acidification rate and oxygen consumption rate were displayed as line grafts. Fasting in mice led to decreased glycolysis and increased OCR by ECAR ratio in bone marrow monocytes, as observed in both mitochondrial and glycolysis stress tests.