A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages
Summary
This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.
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 pathays11,12.
In contrast to M1-like macrophages, M2-like macrophages have decreased glycolysis and increased OXPHOS and are involved in anti-parasitic and tissue repair activities8,13. 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
Representative Results
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 pathways17,18. Understandin…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Ms. Joanna Rocha for editorial assistance. The work was partially supported by the National Institutes of Health (NIH) R01DK118334 (to Drs. Sun and Alaniz) and (NIH) R01A11064Z (to Drs. Jayaraman and Alaniz).
Materials
| 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 |
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