Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Abstract

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

Introduction

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis, including removal of apoptotic cells and remodelling of the extracellular matrix1. These cells are characterized by a strong phenotypic plasticity2 that translates into a many possible activation states, which are also called polarizations. The precise molecular description of these various polarizations is a priority in the field of macrophage biology3. It has been proposed to classify these polarizations using the so-called M1/M2 dichotomy, in ....

Protocol

Human blood samples (LRSC) from healthy, de-identified donors were obtained from EFS (French National Blood Service) as part of an authorized protocol (CODECOH DC-2018–3114). Donors gave signed consent for the use of blood.

1. Media and Buffer Preparation

  1. Prepare the macrophage medium [RPMI glutamax + 10 mM HEPES + 1x non-essential amino acids (NEAA)] and warm it to 37 °C.
  2. Prepare the macrophage medium + 10% human serum from AB plasma (SAB), filter it (0.22 .......

Representative Results

Starting from peripheral blood mononuclear cells (PBMCs) obtained by differential centrifugation, the protocol permits the obtaining of a population of CD14+ monocytes with an assessed purity of more than 98% by flow cytometry (Figure 1). These monocytes are secondarily differentiated toward various polarizations (Figure 2). When a fractionation on gel is chosen, the migration on SDS-page gels is adapted to obtain the .......

Discussion

Because proteomics is a powerful tool to study the expression of different proteins from a whole cell or subcellular compartments, optimization of the cell lysis protocol and digestion of proteins has been addressed by a number of studies. There are three main classes of methods, which include in-gel digestion (digestion of proteins in polyacrylamide gel matrix)17, digestion in solution18 and filter-aided sample preparation19. This last method, at fi.......

Acknowledgements

AM is funded by the Young Group Leader Program (ATIP/Avenir Inserm-CNRS), by la Ligue Nationale contre le Cancer and la Fondation ARC pour la recherche sur le Cancer. We thank Mariette Matondo from the Mass Spectrometry for Biology platform (UTECHS MSBIO, Pasteur Institute, Paris). We thank Lauren Anderson for her reading of the manuscript.

....

Materials

NameCompanyCatalog NumberComments
Hypoxia Working StationOxford OptronixHypoxylab
C6 Flow cytometerBDAccuri C6
UreaAgilent Technologies5188-6435
Formic acid (FA)ARISTAR450122M
R-250 Coomassie blueBiorad1,610,436
Lipopolysaccharide, E.Coli (LPS)Calbiochem437627
2D clean-up kitGE Healthcare80-6484-51
RPMI 1640 medium, glutamax supplementGibco61870044
HEPES 1 MGibco15630-080
MEM Non-Essential Amino Acids (NEAA) Solution 100XGibco11140-035
Phosphate Buffered Saline (PBS) 1XGibco14190-094
Harvard Apparatus column Reverse C18 micro spin columnHarvard Apparatus74-4601
EDTA 0.5 M, pH 8.0InvitrogenAM9260G
NuPAGE Bis-Tris 4-12%Life Technologies SASNP0321 BOX
CD14 Microbeads humanMiltenyi Biotec130-050-201
MACS separation column LSMiltenyi Biotec130-042-401
Macrophage colony-stimulating factor (M-CSF)Miltenyi Biotec130-096-485
Interleukin 4 (IL4)Miltenyi Biotec130-093-917
Interleukin 13 (IL13)Miltenyi Biotec130-112-410
Interferon gamma (INFγ)Miltenyi Biotec130-096-482
CD14-FITC (clone TÜK4)Miltenyi Biotec130-080-701
MACSmix Tube RotatorMiltenyi Biotec130-090-753
Trifluoroacetic Acid (TFA)Pierce28904
Trypsin/Lys-C MixPROMEGAV5073
Complete Mini, EDTA-free Protease Inhibitor cocktailRoche11836170001
Density Gradient Solution (Histopaque 1077)Sigma Aldrich10771-100ML
AccumaxSigma AldrichA7089-100ML
Human Serum from human male AB plasma (SAB)Sigma AldrichH4522-100ML
Bovine Serum Albumin (BSA) solution 30%Sigma AldrichA9576-50ML
Trisma-baseSigma AldrichT1503
GlycerolSigma Aldrich49767
β-MercaptoethanolSigma AldrichM3148
Bromophenol blueSigma Aldrich114405
Sodium Dodecyl Sulfate (SDS) 20%Sigma Aldrich5030
Ammonium bicarbonateSigma Aldrich9830
AcetonitrileSigma Aldrich34888
DithiothreitolSigma Aldrich43819
IodoacetamideSigma Aldrich57670
ThioureaSigma AldrichT8656
CHAPSSigma AldrichC9426
Micro BCA Assay KitThermoFisher23235
5 mL sterile plastic pipetteVWR612-1685
Thermomixer C EppendorfVWR460-0223
Sep-Pak tC18 reverse phase cartridges, 100 mgWatersWAT036820

References

  1. Okabe, Y., Medzhitov, R. Tissue biology perspective on macrophages. Nature Immunology. 17 (1), 9-17 (2016).
  2. Sica, A., Mantovani, A. Macrophage plasticity and polarization: in vivo veritas. The Journal of Clinical Investi....

Explore More Articles

Proteomic AnalysisHuman Macrophage PolarizationLow Oxygen EnvironmentLabel free QuantificationPBMC IsolationCD14 Monocyte IsolationMacrophage Differentiation

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved