PMID- 33616101
OWN - NLM
STAT- MEDLINE
DCOM- 20210319
LR  - 20240226
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 168
DP  - 2021 Feb 7
TI  - A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, 
      and Glycolytic Characterization of Bone Marrow-Derived Macrophages.
LID - 10.3791/61959 [doi]
AB  - 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-gamma, 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 naive 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.
FAU - Eshghjoo, Sahar
AU  - Eshghjoo S
AD  - Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas 
      A&M University Health Science Center.
FAU - Kim, Da Mi
AU  - Kim DM
AD  - Department of Nutrition and Food Science, Texas A&M University.
FAU - Jayaraman, Arul
AU  - Jayaraman A
AD  - Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas 
      A&M University Health Science Center; Artie McFerrin Department of Chemical 
      Engineering, Texas A&M University.
FAU - Sun, Yuxiang
AU  - Sun Y
AD  - Department of Nutrition and Food Science, Texas A&M University; 
      yuxiangs@tamu.edu.
FAU - Alaniz, Robert C
AU  - Alaniz RC
AD  - Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas 
      A&M University Health Science Center; robert_alaniz@tamu.edu.
LA  - eng
GR  - R01 AG064869/AG/NIA NIH HHS/United States
GR  - R01 DK118334/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Video-Audio Media
DEP - 20210207
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
SB  - IM
MH  - Animals
MH  - Biological Assay
MH  - Cell Culture Techniques/*methods
MH  - Cell Fractionation
MH  - *Cell Polarity
MH  - Cell Separation/*methods
MH  - Cells, Cultured
MH  - Energy Metabolism
MH  - Erythrocytes/cytology
MH  - Femur/cytology
MH  - *Glycolysis
MH  - Macrophages/*cytology/metabolism
MH  - Mice, Inbred C57BL
MH  - Phenotype
MH  - Mice
PMC - PMC8118145
MID - NIHMS1683003
COIS- Disclosures The authors have nothing to disclose.
EDAT- 2021/02/23 06:00
MHDA- 2021/03/20 06:00
PMCR- 2021/05/13
CRDT- 2021/02/22 09:18
PHST- 2021/02/22 09:18 [entrez]
PHST- 2021/02/23 06:00 [pubmed]
PHST- 2021/03/20 06:00 [medline]
PHST- 2021/05/13 00:00 [pmc-release]
AID - 10.3791/61959 [doi]
PST - epublish
SO  - J Vis Exp. 2021 Feb 7;(168):10.3791/61959. doi: 10.3791/61959.