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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.
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.
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 ....
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
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 .......
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.......
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.
....Name | Company | Catalog Number | Comments |
Hypoxia Working Station | Oxford Optronix | Hypoxylab | |
C6 Flow cytometer | BD | Accuri C6 | |
Urea | Agilent Technologies | 5188-6435 | |
Formic acid (FA) | ARISTAR | 450122M | |
R-250 Coomassie blue | Biorad | 1,610,436 | |
Lipopolysaccharide, E.Coli (LPS) | Calbiochem | 437627 | |
2D clean-up kit | GE Healthcare | 80-6484-51 | |
RPMI 1640 medium, glutamax supplement | Gibco | 61870044 | |
HEPES 1 M | Gibco | 15630-080 | |
MEM Non-Essential Amino Acids (NEAA) Solution 100X | Gibco | 11140-035 | |
Phosphate Buffered Saline (PBS) 1X | Gibco | 14190-094 | |
Harvard Apparatus column Reverse C18 micro spin column | Harvard Apparatus | 74-4601 | |
EDTA 0.5 M, pH 8.0 | Invitrogen | AM9260G | |
NuPAGE Bis-Tris 4-12% | Life Technologies SAS | NP0321 BOX | |
CD14 Microbeads human | Miltenyi Biotec | 130-050-201 | |
MACS separation column LS | Miltenyi Biotec | 130-042-401 | |
Macrophage colony-stimulating factor (M-CSF) | Miltenyi Biotec | 130-096-485 | |
Interleukin 4 (IL4) | Miltenyi Biotec | 130-093-917 | |
Interleukin 13 (IL13) | Miltenyi Biotec | 130-112-410 | |
Interferon gamma (INFγ) | Miltenyi Biotec | 130-096-482 | |
CD14-FITC (clone TÜK4) | Miltenyi Biotec | 130-080-701 | |
MACSmix Tube Rotator | Miltenyi Biotec | 130-090-753 | |
Trifluoroacetic Acid (TFA) | Pierce | 28904 | |
Trypsin/Lys-C Mix | PROMEGA | V5073 | |
Complete Mini, EDTA-free Protease Inhibitor cocktail | Roche | 11836170001 | |
Density Gradient Solution (Histopaque 1077) | Sigma Aldrich | 10771-100ML | |
Accumax | Sigma Aldrich | A7089-100ML | |
Human Serum from human male AB plasma (SAB) | Sigma Aldrich | H4522-100ML | |
Bovine Serum Albumin (BSA) solution 30% | Sigma Aldrich | A9576-50ML | |
Trisma-base | Sigma Aldrich | T1503 | |
Glycerol | Sigma Aldrich | 49767 | |
β-Mercaptoethanol | Sigma Aldrich | M3148 | |
Bromophenol blue | Sigma Aldrich | 114405 | |
Sodium Dodecyl Sulfate (SDS) 20% | Sigma Aldrich | 5030 | |
Ammonium bicarbonate | Sigma Aldrich | 9830 | |
Acetonitrile | Sigma Aldrich | 34888 | |
Dithiothreitol | Sigma Aldrich | 43819 | |
Iodoacetamide | Sigma Aldrich | 57670 | |
Thiourea | Sigma Aldrich | T8656 | |
CHAPS | Sigma Aldrich | C9426 | |
Micro BCA Assay Kit | ThermoFisher | 23235 | |
5 mL sterile plastic pipette | VWR | 612-1685 | |
Thermomixer C Eppendorf | VWR | 460-0223 | |
Sep-Pak tC18 reverse phase cartridges, 100 mg | Waters | WAT036820 |
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