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 macr....

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 g.......

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. Understandin.......

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