A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Abstract

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-γ, IL-12 & IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host's defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.

This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.

In this paper, we polarized the naïve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

Introduction

Macrophages are one of the most critical cells of the innate immune system M1-like. They are involved in clearing infectious diseases, phagocytosis, antigen presentation, and inflammation regulation². Furthermore, macrophages are required to regulate other immune cells via various cytokines they release³. There is a big spectrum in macrophage phenotypes⁴. Depending on the signals that macrophages are exposed to, they polarize toward different inflammatory and metabolic states⁵. Macrophages manifest metabolic alterations in various diseases, depending on what tissue the macrophages reside⁶. Polarized macrophages have the capability to reprogram or switch their glycolytic metabolism, lipid metabolism, amino acid metabolism, and mitochondrial oxidative phosphorylation (OXPHOS)^7,8. Classically activated M1-like macrophages and alternatively activated M2-like macrophages are the two most studied phenotypes of macrophages³. Non-activated quiescent macrophages are referred to as M0 macrophages. Polarization of M0 macrophages towards an M1-like phenotype can be induced by stimulation of naive BMDMs with bacterial lipopolysaccharide (LPS)⁹. The PI3K-AKT-mTOR-HIF1a signaling pathway can be activated in macrophages in the presence of inflammatory cytokines, interferon-gamma (IFN γ,) or tumor necrosis factor (TNF)¹⁰. M1-like macrophages have increased levels of glycolysis metabolism, decreased levels of oxidative phosphorylation (OXPHOS), producing inflammatory cytokines involved in infectious and inflammatory diseases⁸. On the other hand, polarization towards the M2-like phenotype can be induced by Interleukin (IL)-4, via the JAK-STAT, PPAR, and AMPK pathways, or by (IL)-13 and TGFβ pathays^11,12.

In contrast to M1-like macrophages, M2-like macrophages have decreased glycolysis and increased OXPHOS and are involved in anti-parasitic and tissue repair activities^8,13. BMDMs are a widely used system for the study of macrophages that are derived from bone marrow stem cells. Glycolysis and OXPHOS are the two leading energy production pathways in the cells¹⁴. Based on their microenvironment, BMDMs can choose to use either of these pathways; in some cases, switch from one to another, or use both pathways¹⁴. In this study, we focused on glycolysis metabolism in activated pro-inflammatory macrophages. When the glucose in the cytoplasm is converted to pyruvate and then lactate, the cells produce protons in the medium that cause an elevation in the acidification rate in the surrounded medium of M1-like cells⁵. An extracellular flux analyzer was used to measure the acidification rate of the cell media. Results are reported as Extracellular Acidification Rate (ECAR) or as Proton Efflux Rate.

An optimized quick and easy method to access glycolysis levels in polarized macrophages is essential to determine the glycolytic phenotype, metabolite changes, and the effects of inhibitors/activators and drugs on the polarized macrophages. The method described in this manuscript has been optimized to give information about specific glycolysis factors (Glycolysis, Glycolytic capacity, Glycolytic reserve, and Non-glycolytic acidification), as well as the metabolic reprogramming of glycolytic metabolism. The inhibitor (2DG) that has been used in this study explicitly targets the glycolysis pathway.

This optimized protocol has been modified and improved based on the combination of a published protocol¹⁶, extracellular flux analysis of glycolytic assays of manufacturer's user guides, and direct communication with manufacturer's R&D scientists.

Protocol

Mice were humanely sacrificed according to Assessment and Accreditation of Laboratory Animal Care (AAALAC) and American Association for Laboratory Animal Science (AALAS) guidelines and using protocols approved by the Texas A&M University institutional animal care and use committee (IACUC).

1. Mice bone marrow harvest and culture of BMDMs

1. Sacrifice mouse (6-10 weeks of age C57Bl/6 mice were in this protocol) and lay it on its ventral side, cut the skin and peritoneal layer and gently peel off the legs.
   NOTE: Use CO₂ gas exposure to euthanize the mouse.
2. Separate both hind legs from the hip down, being careful not to cut the bone.
3. Place the whole leg in an empty 50 mL conical tube (with feet facing up to have an easy grip to pull out later) on ice and proceed with harvesting both legs from the mouse.

2. Femur exposure

NOTE: Perform the following steps in a biosafety cabinet.

1. Harvest the femur by cutting off the tibia from each leg and remove as much tissue surrounding the femur as possible with scissors and laboratory paper.
2. Place harvested, "cleaned" femurs in a 10 cm plate containing a piece of laboratory paper saturated with tissue culture (TC) medium or PBS. Place them on ice.
3. Continue harvesting femurs and removing tissue from all femurs before proceeding to the flushing stage (Figure 1A).

3. Marrow flush

1. To flush marrow from femurs, use a 3 mL syringe filled with TC medium or PBS with a 23G needle. Fill the syringe before exposing the marrow.
2. Use scissors to expose the marrow by cutting the very end of the femur at both epiphyses.
3. Insert needle tip into the femur and gently flush marrow out into a 10 cm dish.
4. Run the needle through the entire length of the femur, and flush until the bone color turns white. Usually, most marrow can be flushed out with 2-3 mL of media.
5. Flush all femurs and pool bone marrow in the dish. Use a needle to break up any visible clumps. Strain marrow into a 50 mL conical tube (Figure 1A).

4. RBC lysis

1. Spin marrow at 190 x g for 10 min. Aspirate the supernatant.
2. Resuspend the pellet in 4 mL of ACK lysis buffer with a pipette. Allow RBC lysis buffer to work for 5 min at room temperature.
3. Add 4 mL of TC medium RPMI-C 10% (RPMI 1640 -GlutaMAX) supplemented with 2-mercaptoethanol, gentamicin, streptomycin, and 10% FCS to the marrow suspension and spin at 1300 x g for 10 min.
4. Strain again to remove RBC debris and re-suspend in a small volume of RPMI-C 10% for counting.
5. Count cells with a cell counter (Figure 1B). A Vi-Cell Counter was used to determine the count and viability of cells in the suspension.

5. Plating and culture

1. Add 10 mL of RPMI-C 10% + 10 ng/mL M-CSF (Macrophage Colony Stimulating Factor, an essential regulator of monocyte/macrophage proliferation, differentiation, and survival) to as many 10 cm plates as desired.
2. Add an appropriate volume of counted cells so that each 10 cm plate contains 1 x 10⁶ cells. Put the plates in a 37 °C incubator (defined as day 0).
3. On day 3, gently add 5 mL of fresh RPMI-C 10% + 10 ng/mL M-CSF to each plate.
   NOTE: On day 7, BMDMs should be ready for testing (Figure 1C).

6. Harvest from plates

1. Use a light microscope to confirm that most cells have adhered to plates.
2. Gently aspirate media. Then add 3 mL of PBS and gently swirl the plate. Aspirate this well to remove any remaining non-adherent cells.
3. Add 7-10 mL of cold PBS to the plate, use a P1000 pipette to wash the bottom of plates, and harvest all remaining cells into a collection tube.
   NOTE: Keep tubes on ice as macrophages are very tightly adherent and will adhere to the inside of the tube. If the cells kept cold, they would be less tightly adherent.
4. Centrifuge, count, and plate cells for experiments (Figure 1D). Using Flow cytometry, resulting cells should be >95% positive for CD11b and F4/80. (macrophage polarization was determined by staining with M1-like markers of CD38, TNF-a, and MCP-1 and M2-like marker of CD206.
   NOTE: Perform steps 6.1-6.3 in the biosafety cabinet and perform step 6.4 on the benchtop. Maintain aseptic techniques throughout the procedure.

Figure 1: Graphical workflow of mouse bone marrow culture of BM-Derived Macrophages. (A) Leg harvest, Femur exposure, and marrow flush; (B) RBC Lysis; (C) Plating and culture; (D) Cell harvest from the plates. Please click here to view a larger version of this figure.

7. The day before the metabolic flux analyzer assay: seeding and polarization of the cells for the glycolytic test

1. Warm up the Metabolic Flux Analyzer to 37 °C by turning on the instrument.
2. Hydrate a cartridge by adding 200 µL of a Calibrant Solution and incubate the cartridge in a non-CO₂ incubator overnight (Figure 2A). The humidity of non-CO₂ incubator is not important for cartridge hydration.
   1. An hour prior to the experiment, dip the plate a few times up and down, which will help to remove air bubbles.
3. Design the plate map on the software in the default glycolysis stress test-acute injection, by following the instruction of the test.
   1. Click on the software icon, and then click on Glycolysis stress-acute injection test. On the group definition icon, generate group names.
4. There are five measurement cycles with a duration of 18 minutes and four injections. Change the injection of port A to Glucose, port B to Oligomycin, Port C to Rotenone and antimycin A (Rot/AA), and Port D to 2DG.
5. Re-suspend the cells in RPMI-C 10% medium and seed 50k cells per well except for the four edges of the plate (A1, A12, H1, and H12; Add media only, no cells) in a Metabolic Flux Analyzer microplate to a final volume of 100 µL. Normally a minimum of 40k cells is required to conduct this assay.
6. Allow the cells to sit at room temperature for 45 min to avoid the edge effect of the cells. The edge effect is when the medium from around the perimeter of the plate evaporates partly, which causes volume and concentration changes and reduces cell viability.
7. Add 10 ng/mL LPS to polarize the naïve macrophages towards M1-like cells and add 20 ng/ml of IL-4 to polarize them towards M2-like cells. Use at least 3 to 6 wells per condition (Figure 2B).
8. Check the cells under the microscope and place the plate in an incubator at 37 °C and 5% CO₂ for 24 hours.

Figure 2: Graphical demonstration of seeding and polarization of the cells. (A) Extracellular flux analyzer set up and cartridge hydration; (B) Polarization of the cells and overnight incubation. Please click here to view a larger version of this figure.

8. Day of the assay: XF Medium and compound preparation

1. Complement 100 mL of XF RPMI (pH 7.4) assay medium with 2 mM glutamine.
2. Filter-sterilize the media using a 0.2 µm vacuum filter system.
3. Place the assay media in a 37^°C water bath for 20 min.
4. Remove the plated cells from the 37 °C, 5% CO₂ incubator. Wash the cells with assay media twice and replace the previous media with assay media to the final volume of 180 µL.
5. Use a microscope to make sure that all the wells have confluent cells and mark any wells that have any scratches from pipetting. If there are any scratches, remove that plate before analyzing.
6. Position the cell-containing plate in a non-CO₂ incubator for 45 min (Figure 3A).
7. Using the compounds and the assay media to make stock solutions of Glucose (100 mM), Oligomycin (100 µM), Rot/AA (50 µM), and 2DG (500 mM) (Table 1).
8. Make a 10x injection mixture of each compound using assay media (Table 2).

Injection Stocks (Provided in the kits)	Add Complete assay media (mL)	Final Stock concentration (μM)
Glucose	3	100K
Oligomycin	0.72	100
2-DG	3	100k

Table 1. Injection stocks

Ports on the Cartridge	Stock solutions	Add stock volume	Add assay media	Final concentration of injections (10x)	Add this volume to designated port (μL)	Final concentration after injection in each well
A	Glucose (100 mM)	3000 μL + 0 μL		100 mM	20	10 mM
B	Oligomycin (100 μM)	300 μL + 2700 μL		10 μM	22	1.0 μM
C	Rotenone/ Antimycin A (50 μM)	300 μL + 2700 μL		5 μM	25	0.5 μM
D	2-DG (500 mM)	300 μL + 0 μL		500 mM	28	50 mM

Table 2. Final Injection Concentrations

9. Day of the assay: Running the acute glycolytic test on polarized macrophages

1. Open the saved Glycolysis stress assay (Acute injection) template from the software. The default Acute Glyco-Stress Test has 3 minutes of mix and measurement before each injection.
2. Check the template and the assay details, and when ready, click on Run and follow the instruction of the default assay. However, all parameters can be customized.
3. Remove the Sensor Cartridge from the non-CO₂ incubator, remove the lid, and insert in the instrument in a way that the A1 well of the cartridge plate falls into the top left corner of the insertion panel of the machine. Usually, calibration takes between 20 to 45 min.
4. After finishing the calibration, the device will eject the plate containing the calibrant solution and will hold the sensor cartridge. Remove the calibrant containing the plate.
5. Remove the cell plate from the non-CO₂ incubator, remove the plate lid, and insert it in the machine. Click on Run (Figure 3B).
6. When the assay is done, the machine will eject the cell plate and the sensor cartridge.
7. Remove the media from the plate and freeze it at -20 °C for further normalization.
8. Use the commercial cell proliferation assay kit (e.g., CyQUANT) for normalizing the cells.
9. Add 1 mL of Compound B or lysis buffer to 19 mL of nuclease-free distilled water.
10. Add 100 µL of Compound A or GR working solution to the abovementioned solution.
11. Make sure the cells in the plate are thawed and then add 200 µL of the solution to each well.
12. Incubate for 5 min at room temperature (RT).
13. Measure the fluorescence in 480 nm excitation and 520 nm emission wavelengths using a plate reader.
14. Normalize the cells on the normalization panel of the software.
15. Normalize cells based on naïve macrophages cell count (Figure 3C). Consider the average of the naïve macrophages as 1 (by dividing the cell number of each well by the average cell number of naïve macrophages) and apply them to all macrophages.

Figure 3: Day of the assay: medium and compound preparation and running the assay. (A) Cells preparation for assay; (B) Compounds preparation, calibration, and running the assay; (C) Normalization and data analysis. Please click here to view a larger version of this figure.

Representative Results

Glycolysis and mitochondrial oxidative phosphorylation are the two major ATP production pathways in the cells (Figure 4A). Some cells have the capability to switch between these two pathways to meet their energy demands. The conversion of glucose to pyruvate in the cytoplasm is called glycolysis. Pyruvate has two fates; it will either get converted to lactate or further metabolized through the TCA cycle and eventually through the electron transport chain (ETC) to produce more ATP. In order to obtain the best understanding of the glycolytic parameters of cells, we minimize the OXPHOS pathways by using oligomycin, which is an inhibitor for mitochondrial ATP synthase. We also inject Rot/AA to completely shut down the ETC to assess the maximal glycolytic capacity and the compensatory glycolysis in the cell (Figure 4A). Glucose is the primary fuel of the glycolysis. Since the XF assay medium does not have any glucose or glutamine, the first three measurements in the assay will be an indicator of non-glycolytic acidification rate (Figure 4B,C), which is indicative of acidification but not related to the conversion of glucose to lactate. After injection of glucose from port A, increased ECAR levels are indicators of glycolysis rates (measurements 4,5 & 6). Next, by injection of oligomycin from port B and injection of Rot/AA from port C, the ETC is inhibited, and increased amounts of ECAR are an indicator of glycolytic capacity and the compensatory glycolysis rates of the cells (Figure 4B,C). Compensatory glycolysis rates of BMDMs demonstrates the cellular energy management capability under mitochondrial stress conditions. In other words, this parameter indicates compensation for energy demand when mitochondrial respiration is inhibited. The last injection is 2 deoxyglucose or 2DG from port D which is a competitive inhibitor of the glucose.

In Figure 4B,C an alternative calculation, especially if there are big errors between 3 measurements, is that one can measure the last data point before each injection and prevent unnecessary errors and variations in the data.

Figure 4: Energy production in the cell and glycolytic parameters. (A) schematic view of the two most important energy production pathways in the cell; Glycolysis (left) and mitochondrial oxidative phosphorylation (right). Glycolysis is the conversion of glucose to pyruvate. XF analyzer can detect the protons that are produced by conversion of pyruvate to lactate as ECAR (mpH/min) levels. Inhibition of the ATP synthase followed by inhibition of complex I and II the in mitochondrial electron transport chain will eliminate the ATP production and proton efflux through OCR. (B) calculation of glycolytic parameters. (C) Glycolytic function parameters after each compound injection. Please click here to view a larger version of this figure.

Generally, polarized macrophages have more glycolytic activities compared to naïve M0 macrophages. LPS induced M1-like macrophages possess the highest glycolytic activity. Although polarized macrophages have more distinct separations in their OCR spare respiratory capacity (16), which has not been shown here, their glycolytic metabolism is also wholly distinguishable. It is important to note that an increase in ECAR in LPS induced M1-like polarized BMDMs is not a definite characteristic for other types of M1-like polarized macrophages (such as LPS + INF-γ or PAMP induced M1-likes macrophages), and they may not increase or change the ECAR without glycolytic stress. As expected, in the first three measurements, which is the indicator of the non-glycolytic activity, polarized macrophages do not show a significant difference because the media do not have any sources of glucose or pyruvate (Figure 5). After injection of glucose, polarized BMDMs indicate higher levels of glycolysis than naïve BMDMs, and M1-like BMDMs demonstrate the highest levels of glycolysis compared to M0 and M1-like.

Typically, glycolytic metabolism is the preferred ATP production pathway used by M1-like macrophages, and OXPHOS is the main ATP production pathway for M2-like macrophages. After the injection of Oligomycin, the ATP synthase complex in the mitochondrial electron transport chain of BMDMs will shut down; thus, the cells will start to rely on glycolysis to meet their energy demands. Since glucose is present in the media, the glycolytic capacity of the polarized BMDMs will be comparable. Again, LPS-induced M1-like BMDMs will have the highest glycolytic capacity. M2-like and M0 will have lower glycolytic capacities, respectively (Figure 5). Injection of Rot/AA will inhibit the Complex I and III of the mitochondrial ETC and completely shut down the OXPHOS, and there will be a slightly higher increase in the ECAR levels, which is an indicator of compensatory glycolysis. Again, M1-like BMDMs will have the highest ECAR levels in this step; finally, 2DG, a competitive inhibitor of glucose and negative control for glycolysis, will completely shut down the Glycolysis pathway.

Figure 5: Glycolytic functions of naïve M0 and polarized M1-like (LPS-induced) and M2-like (IL4-induced) BMDMs. Glycolytic parameters of polarized macrophages indicated as ECAR (mpH/min). (A) Non-glycolytic acidification rate, Glycolysis, Glycolytic capacity, and compensatory glycolysis as ECAR (mpH/min) in M0, M1-like, and M2-like BMDMs. Injections of the ports are as follow → port A: Glucose, Port B: Oligomycin, Port C: Rotenone plus antimycin A, and port D: 2 Deoxy Glucose (B) Bar graphs of each parameter for M0, M1-like, and M2-like BMDMs. Data shown are from 4-6 culture wells per experiment. Measurements are based on means + SEM. Statistical significance between groups is based on one-way ANOVA with Tukey's multiple comparison test at "*" p < 0.05, significance at "**" p < 0.01, significance at "***" p < 0.001, significance at "****" p < 0.0001.Error bars are derived from standard deviation. Please click here to view a larger version of this figure.

Discussion

As mentioned earlier, the extracellular flux analyzer machine can provide real-time information about two major energy-producing pathways of the cells by measuring OCR (oxygen consumption rate), an indicator of mitochondrial OXPHOS activity, and ECAR (extracellular acidification rate) which is an indicator of glycolysis. Macrophages can use both pathways, depending on their microenvironment. They can also switch their energy production pathways^17,18. Understanding the macrophages' energetic states and their responses to different drugs, cytokines, inhibitors, activators, etc. will provide a better understanding of the metabolic states of these cells. Since glycolysis is one of the most critical pathways that get activated in M1-like types of macrophages, real-time glycolysis related information can help track the changes of M1-like polarized BMDMs in different in vitro conditions¹⁹. The extracellular flux analysis of the ATP real-time rate assay is highly regarded as a way to assess the ATP production of the polarized BMDMs²⁰. The protocol presented herein provides technical details and approaches with a visual demonstration of the workflow to serve as a comprehensive protocol that can be adapted to experimental needs.

This assay provides accurate measurements of glycolytic levels for basal conditions and compensatory glycolysis following mitochondrial inhibition. It is important to note that some of the acidification in extracellular media can have a mitochondrial source²¹. The Krebs cycle or TCA cycle produces CO₂ that can acidify the media through its reaction with water molecules²². When the mitochondrial activity is inhibited, the acidification rates are indicators of lactate accumulation in the media. The advantage of the glycolytic stress test is the injection of glucose in a medium that does not have any glucose or pyruvate sources to assess glycolysis levels before and after the treatment of glucose in the media.

On the other hand, the glycolytic rate assay provides specific information about the distinct sources of glycolysis by blocking the mitochondrial activity. In other words, glycolytic proton efflux can be calculated by subtracting OXPHOS proton efflux from total proton efflux. In this protocol, we combined glycolytic assays in one assay and maximized the glycolytic data to obtain glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, and non-glycolytic acidification results. These parameters will give a better understanding of the metabolic states and glycolytic phenotype of the cells. With an optimized quick and easy normalization method, it would be possible to get more accurate information about glycolytic metabolism and metabolic reprogramming^21,22,23.

It is important to note that although the implication of glycolytic reserve does not change in the new combined system, the calculation scheme in the method has been altered slightly. In the combined system (Figure 4B), the glycolytic reserve is estimated by Avg. ECAR (10,11,12)-Avg. ECAR (4,5,6). However, in non-combined methods, the glycolytic reserve is calculated by Avg. ECAR (7,8,9)-Avg. ECAR (4,5,6) formula. Both calculations provide very similar results and reflect the glycolytic reserve.

Furthermore, it is worth mentioning that there are different metrics of extracellular acidifications in extracellular flux analyzers. The results of the glycolytic assays in extracellular flux analyzers can be analyzed based on ECAR (mpH/min), PPR (pmol H+/min), and PER (pmol H+/min). There are advantages and disadvantages, but generally, ECAR is the most typical way of displaying extracellular acidification data.

Our lab studies the role of microbiota metabolites on immune cells. Since macrophages are one of the key components of the immune system in chronic inflammatory diseases such as atherosclerosis^23,24,25, we are interested in studying the role of microbiome metabolites on the polarization of macrophages, especially inflammatory polarized macrophages that have been induced by different proatherogenic signals such as saturated fatty acids, modified LDLs, and harmful gut microbiota-derived or dependent metabolites. We confirm the polarization of the BMDMs by M1-like and M2-like surface and intracellular markers using flow cytometry and qPCR. We consider the extracellular flux assays as functional readouts in the studies. We perform complementary studies by measuring the non-real-time glycolysis factors with a lactate assay.

LPS induced M1-like polarization or LPS + IFNγ induced M1-like polarization are the most common classic M1-like activation way in macrophages. In M1-like polarization, adding IFNγ to LPS or increasing LPS concentration will increase the reduction of spare respiratory capacity in the mitochondrial electron transport chain. IFNγ is known to induce an M1-like phenotype, but, usually, IFNγ by itself is not enough and requires additional TLRs agonists to induce the phenotype. But this is dependent on the diseases and M1-like polarization concerning a specific condition. For example, IFNγ- induced M1-like macrophages cannot produce NO and inflammatory cytokines similar to LPS or LPS/ IFNγ induced macrophages²⁶.

Drugs that prevent macrophages from inflammatory polarization have the potential to prevent and control atherosclerosis. Understanding the metabolic pathways, energetics, and phenotypic characteristics of the M1 macrophages is essential for studying the role of different endogenous and exogenous drugs. Glycolysis is the dominant energy-producing pathway in M1-like macrophages^24,27.

This simplified study focuses on only glycolytic energetic states of the polarized BMDMs. Doses used in this paper are built on the manufacturer's recommendation and make the experiment much easier to follow. Also, most of the compounds used in this study are provided in the standard kit from the manufacturer; this helps to save time and enhance the consistency of the experiments. It is essential to know that slight differences in compound doses, cell numbers, and incubation times can affect the experiment results. Also, each experimenter should run a cell titration, dose-response, and kinetic analysis for their particular cell type and conditions to understand how those conditions perform on the extracellular flux analyzer.

One should note that during the media change, washing, and normalization steps, some cells may come off by pipetting or fluid pressure. The confluency of the cells is always detectable under the microscope. Those wells need to be excluded from the study if they have any signs of scratches or depletion of cells.

We use 96 well microplates with a minimal number of cells per well, allowing positive and negative controls, as well as different conditions to be tested in one plate; thus, this assay is very time-saving and cost-efficient for extracellular flux analysis. This study has been optimized for BMDM, which are different from tissue-resident macrophages, peritoneal macrophages, and macrophage cell lines.

While in this protocol we primarily focused on the application of extracellular flux analysis in the pro-inflammatory state associated glycolysis, extracellular flux analysis can also be used to assess mitochondrial function characteristics such as total respiration, basal mitochondrial respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity. Mitochondria play an important role in macrophage metabolic reprogramming. Extracellular flux analyzers have been used to assess mitochondrial stress and fatty acid oxidation by measuring the oxygen consumption rate of the cells¹⁶.

In conclusion, here, we have provided a comprehensive protocol for isolation, culture, polarization, and glycolytic functional analysis of BMDMs. Detailed step-by-step procedures and visual demonstrations were provided for all steps. We hope this protocol will help the investigators to streamline their analyses and to assess the glycolytic function of BMDMs with high quality and efficiency.

Materials

Name	Company	Catalog Number	Comments
23G needles	VWR	BD305145
2-mercaptoethanol	Life Technologies	21985023
50ml Conical Tube	VWR	21008-951
ACK lysis buffer	Thermo Fisher Scientific	A1049201	It can be lab-made
Agilent Seahorse XF glycolysis stress test kit	Agilent Technologies	103020-100
Agilent Seahorse XF Glycolysis Stress Test Kit User Guide	Agilent Technologies	103020-400
Agilent Seahorse XF Glycolytic Rate Assay Kit	Agilent Technologies	103344-100
Agilent Seahorse XF Glycolytic Rate Assay Kit User Guide	Agilent Technologies	103344-100
Alexa Fluor 488 anti-mouse CD206 (MMR) Antibody	BioLegend	141710
anti-mouse CD11b eFluor450 100ug	eBioscience	48-0112-82
BD 3ML - SYRINGE	VWR	BD309657	Other syringes are acceptable too
Cell counter-Vi-CELL- XR Complete System	BECKMAN COULTER Life Sciences	731050	Cells can be manually counted too
Cell Strainer-70µm	VWR	10199-656
CyQUANT Cell Proliferation Assay Kit	Thermo Fisher Scientific	C7026
F4/80 monoclonal antibody (BM8) pe-Cyanine7	eBioscience	25-4801-82
Fetal Bovine Serum	Life Technologies	16000-044
Flow cytometer: BD LSFRFortessa X-20	BD	656385
Kim Wipes	VWR	82003-822
LPS-SM ultrapure (tlrl-smpls) 5 mg	Invivogen	tlrl-smlps
MCSF	Peprotech	315-02
Murine IL-4	Peprotech	214-14
PE Rat Anti-Mouse CD38	BD Biosciences	553764
Penicillin-Streptomycin (10,000 U/mL)	Life Technologies	15140122
Petri Dish 100mm x 15 mm	Fisher Scientific	F80875712
RPMI, Glutamax, HEPES	Invitrogen	72400-120
Seahorse Calibrant Solution	Agilent Technologies	103059-000
Seahorse XF 200mM Glutamine Solution	Agilent Technologies	103579-100
Seahorse XF Glycolytic Rate Assay Kit	Agilent Technologies	103344-100
Seahorse XFe96 FluxPaks	Agilent Technologies	102416-100
XF Glycolysis Stress Test Kit	Agilent Technologies	103020-100
XF RPMI Medium, pH 7.4 without phenol Red	Agilent Technologies	103336-100