Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage’s metabolism and phenotype.

The overall goal of this extracellular flux analysis is to assess the metabolic characteristics of naive M1 and M two polarized bone marrow derived macrophages. This method can serve as a framework for investigating how particular cytokines, compounds, or geno codes affect the metabolic phenotype of macrophages. The main advantage of this technique is that it requires only small amount of macrophages.

Moreover, it measures all relevant glycolysis and mitochondrial parameters in one single assay. Though this method provides insight into the metabolic phenotype of bone marrow derived macrophages, it also can be applied to all the types of macrophage or immune cells. We had the idea to publish this method in J since we frequently receive requests from other scientists to assist them with their metabolic access cellular flux assays On day eight of culture, verify the presence of mature macrophages by visual inspection and incubate the mature bone marrow derived macrophages in 10 milliliters of citrate saline for five minutes.

A 37 degrees Celsius when the cells have detached. Wash the culture with 10 milliliters of PBS and count the number of viable cells. Then seed five times 10 to the fourth cells per well.

In an XFE 96 cell culture, microplate in 100 microliters of culture medium per well, holding the pipette at an angle about halfway down the side of each well. To dispense the cells, add medium alone to the background correction wells A one, A 12, H one, and H 12. Then allow the cells to settle at room temperature in a cell culture hood for one hour.

After confirming adherence culture, the cells in a 37 degree Celsius 5%carbon dioxide incubator. Three hours after plating, stimulate between four to six wells of cells per condition as appropriate for 24 hours. To polarize the bone marrow derived macrophages on the day before the extracellular flux assay, place the sensor cartridge upside down next to the utility plate and fill each plate well with 200 microliters of crin solution.

Next, lower the sensor cartridge onto the utility plate to submerge the sensors in the Cain solution and verify that the Cain solution level is high enough to keep the sensor submerged. Then place the cartridge in a non-carbon dioxide 37 degrees Celsius incubator overnight. The next day, gently remove the supernatant from the polarized macrophages and wash the cells with 100 microliters of freshly prepared assay.

Medium per well. Add 180 microliters of assay medium to each well, and use the microscope to verify that the cells were not washed away. If the cells are still attached, place the plate in the 37 degree Celsius non-carbon dioxide incubator for one hour prior to the assay run while the cells are incubating.

Prepare 10 x injection compound mixtures as designated in the table and warm the mixtures to 37 degrees Celsius. Then load the sensor cartridge with the appropriate volumes of compound and ports A, B, C, and D using the provided loading guides to characterize the bioenergy of the polarized macrophages by extracellular flux assay. Use the assay wizard to create an assay template with two minute mix and three minute measure times, and at least three mix and rate measurement loops before each of the four injections and after the last injection.

Then start the assay and follow the instructions as indicated by the apparatus. Loading the cartridge plate with the hydrated probes to allow calibration by the apparatus and the cell plate. When instructed, at the end of the assay, carefully discard all of the assay medium and store the analyzed cell culture microplate at minus 20 degrees Celsius until normalization.

To normalize the cells using the cell proliferation assay kit, thaw the plate and incubate the cells in 200 microliters of freshly prepared cell lysis buffer per well for five minutes at room temperature. Finally, measure the fluorescence with about 480 nanometer excitation and about 520 nanometer emission maxima. Using the acquired fluorescence measurements to calculate the ratio in which the average cell count of all naive macrophage wells is set at one, then export these ratios into the seahorse wave analysis software.

To normalize the data, an extracellular flux assay will typically yield extracellular acidification rate or ECAR and oxygen consumption rate, or OCR plots as demonstrated in this representative figure after the injection of glucose, the observed increase in ECAR represents the glycolysis rate. The additional increase observed in the ECAR after a TP synthase inhibition with all liga mycin provides further information about the glycolytic reserve and capacity. When analyzing the OCR values, CIN injection facilitates the calculation of the oxygen consumption used for mitochondrial A TP synthesis, FCCP uncouples mitochondrial respiration.

The corresponding OCR measurements yield data about the maximal and spare respiratory capacity rot known and antimycin a injection, however, block mitochondrial complexes one and three with the residual OCR representing the non mitochondrial oxygen consumption. Macrophage activation with LPS induces an increased glycolytic metabolism. With the differences between LPS and IL four treated macrophages becoming even more apparent when the oxygen consumption rates are observed.

Indeed, while the maximal oxidative metabolism is highly suppressed in LPS treated macrophages IL four induces a robust respiration in M two macrophages. Once mastered, the extracellular flu assay can be completed in two hours if it's performed properly. While attempting this procedure, it's important to remember to inject vet together with FCCP to fuel maximal respiration upon uncoupling.

After this technique, additional assays like Eliza can be performed to assess efficient macrophage polarization. This technique paved the way for researchers from the field of immunology to explore the metabolic characteristics of a wide range of immune cells. After watching this video, you should have a good understanding of how to perform extracellular flux analysis to assess the metabolic phenotype of macrophages.