Richard Ouedraogo is supported by a grant from the Minist&#232;re de la Sant&#233; (PHRC 2010). We thank Laurent Gorvel, Christophe Flaudrops and Nicolas Amstrong for technical assistance.

In the present protocol, the whole-cell MALDI-TOF technique is used to obtain a mass spectrum considered as a fingerprint of the sample. A bioinformatic analysis allowed the comparison and the classification of these fingerprints. There were three main parts in this protocol:
<ol>
	<li>The preparation of the biological samples: control macrophages and macrophages stimulated with different agonists.</li>
	<li>The analysis of each type of sample with technical replicates by whole-cell MALDI-TOF MS.</li>
	<li>The bioinformatics analysis of raw data.</li>
</ol>
Prepare sterile solutions for cell isolation and culture. Prepare and store all reagents at 4 &#176;C
1. Preparation of Human Monocytes
<ol>
	<li>Prepare cell culture medium. Add 55 ml&#160;of human serum AB+ or fetal bovine serum (FBS) and 5 ml&#160;of the antibiotic solution (penicillin at 10,000 UI/ml and streptomycin at 10,000 &#956;g/ml; final concentration of 100 UI/ml&#160;for penicillin and 100 &#956;g/ml&#160;for streptomycin) to RPMI 1640 medium (500 ml).
	<ol>
		<li>Isolate peripheral blood mononuclear cells (PBMCs) from leukopacks (leukocyte concentrates).
		<ol>
			<li>Prepare 50&#160;ml&#160;tubes containing 15 ml&#160;Ficoll. Dilute the blood in saline (vol/vol, 1/10). Deposit 30 ml&#160;diluted PBMCs on Ficoll as previously described26.</li>
			<li>Centrifuge at 700 x g for 20 min. Recover PBMCs at the interface between Ficoll (density of 1.077 g/ml) and diluted plasma. Dilute PBMCs in culture medium and centrifuge at 300 x g for 5 min.</li>
		</ol>
		</li>
		<li>Prepare CD14+ monocytes from PBMCs using magnetic beads coated with anti-CD14 antibodies. 
		Note: Keep products and cells at 4 &#176;C until monocyte obtention.
		<ol>
			<li>Prepare running buffer consisting of phosphate-buffered saline (PBS) pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA).</li>
			<li>Gently dissociate pelleted PBMCs (107 cells per assay) into 80 &#956;l&#160;of running buffer.</li>
			<li>Add 20 &#956;l&#160;CD14 MicroBeads to PBMCs. Mix and incubate PBMCs for 15 min. Wash PBMCs with 5 ml&#160;of running buffer and centrifuge at 300 x g for 5 min. Gently dissociate pelleted PBMCs into 500 &#956;l&#160;of running buffer for 108 PBMCs. 
			Note: use 500 &#956;l&#160;of running buffer for PBMC concentrations lower than 108 PBMCs.</li>
			<li>Proceed to magnetic separation. 
			Note: for less than 2 x 108 PBMCs, use MS column; for more than 2 x 108 PBMCs and less than 2 x 109 PBMCs, use LS column.</li>
			<li>Rinse precolumn and column with running buffer (500 &#956;l&#160;for MS column or 3 ml&#160;for LS column). Add PBMCs into the precolumn (wait for unlabeled cells to pass through the column). Rinse 3 times with running buffer (500 &#956;l&#160;for MS column or 3 ml&#160;for LS column). Remove the precolumn.</li>
			<li>Place the column on 15&#160;ml&#160;tubes. Add running buffer (1 ml&#160;for MS column or 5 ml for LS column) on the column and elute CD14+ cells by applying a pressure on the column.</li>
			<li>Centrifuge the eluate at 300 x g for 5 min. Discard the supernatant. Wash monocyte pellet with 10 ml&#160;of culture medium. 
			Note: Analyze the purity of monocyte preparation using anti-CD14 antibodies and flow cytometry (classically higher than 95%).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Differentiation of monocytes into macrophages.
	<ol>
		<li>Incubate monocytes (106 monocytes per well in 6&#160;well plates) in 3 ml&#160;of culture medium containing 10% human serum AB+ at 37 &#176;C. After 4 days, replace the culture medium containing human serum by 3 ml&#160;culture medium containing 10% FBS for 3 additional days.</li>
		<li>Identify the obtained cell population as monocyte-derived macrophages (MDMs) by flow cytometry (see Figure 1).
		<ol>
			<li>Replace the culture medium by 3 ml&#160;PBS. Scrape MDMs with a rubber policeman and collect MDM suspensions. Centrifuge at 400 x g for 5 min. Add PBS containing 2% BSA to pelleted MDMs and gently agitate cell suspension. Adjust the cell concentration (106 MDMs in 200 ml&#160;PBS) and incubate at 4 &#176;C.</li>
			<li>Add 10 ml&#160;of anti-CD14 antibodies and 10 ml&#160;of anti-CD68 antibodies to MDM suspension and incubate at 4 &#176;C for 20 min in the dark. 
			Note: these antibodies must be labeled with two different fluorochromes consistent with flow cytometry analysis. For example, anti-CD14 antibodies may be conjugated with phycoerythrin coupled (for monocytes staining) and anti-CD68 antibodies with Alexa Fluor 647 coupled (for macrophages staining).</li>
			<li>Centrifuge MDMs at 400 x g for 5 min. Remove supernatants. Gently dissociate the cell pellet and incubate MDMs in 250 ml&#160;3% paraformaldehyde (PFA) for 15 min at room temperature. Centrifuge PFA-fixed MDMs at 400 x g for 5 min. Wash MDMs with 3 ml&#160;PBS and centrifuge MDMs at 400 x g for 5 min. Gently dissociate MDM pellet in 400 ml&#160;PBS.</li>
			<li>Analyze the differentiation of monocytes (that express CD14 but not CD68) into monocytes (that express CD68 but not CD14). Note: classically more than 95% of cells are MDM.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>18 hr&#160;stimulation of MDMs.
	<ol>
		<li>Replace the culture medium of adherent MDMs by fresh 3 ml&#160;culture medium (containing 10% FBS).</li>
		<li>To induce a M1 or M2 polarization, use the following human recombinant cytokines: IFN-&#947; and TNF for M1 polarization, and IL-4 for M2 polarization. M1 macrophages are also obtained by stimulation with LPS. Note that stock-solutions of cytokines are conserved at -80 &#176;C and that the stock-solution of LPS is conserved at -20 &#176;C.
		<ol>
			<li>Dilute the stock-solution of IFN-g&#947; (1 mg/ml) at 1/100 in culture medium to obtain a dilution of 10 ng/ml. Add 6 &#956;l of this IFN-&#947; dilution to MDMs to obtain a final IFN-&#947; concentration of 20 ng/ml.</li>
			<li>Dilute the stock-solution of TNF (1 mg/ml) at 1/100 in culture medium to obtain a dilution of 10 ng/ml. Add 6 &#956;l of this TNF dilution to MDMs to obtain a final TNF concentration of 20 ng/ml.</li>
			<li>Dilute the stock-solution of IL-4 (1 mg/ml) at 1/100 in culture medium to obtain a dilution of 10 ng/ml. Add 6 &#956;l of this IL-4 dilution to MDMs to obtain a final IL-4 concentration of 20 ng/ml.</li>
			<li>Add 3 &#956;l of the stock-solution of LPS (1 mg/ml) to MDMs to obtain a final LPS concentration of 1 &#956;g/ml.</li>
		</ol>
		</li>
		<li>To stimulate MDMs with bacteria, use heat-killed bacteria. Wash living bacteria with PBS and heat them at 95 &#176;C for 1 hr. Use Orientia tsutsugamushi (strain Kato (CSUR R163), Mycobacterium bovis (Bacillus Calmette-Gu&#233;rin, BCG CIP strain 671203) and Coxiella burnetii (Nine Mile in phase I) were because these pathogens are known to infect macrophages. Thus, prepare a O. tsutsugamushi suspension at 109 bacteria/ml. Add 50 &#956;l to MDMs . Repeat the same step for M. bovis and C. burnetii.</li>
	</ol>
	</li>
	<li>Preparation of biological samples for MALDI-TOF MS.
	<ol>
		<li>Wash stimulated MDMs with PBS without Ca2+ or Mg2+ (cells may agglutinate in the presence of Ca2+ or Mg2+). Scrape MDMs with a rubber policeman and collect MDM suspensions. Centrifuge MDMs. Wash again MDMs in PBS without Ca2+ or Mg2+ at 400 x g for 5 min to discard FBS contamination.</li>
		<li>Adjust the cell concentration (2 x 106 MDMs per assay). Centrifuge MDMs at 400 x g for 5 min and discard supernatants. Collect cell pellets in 20 &#956;l of PBS without Ca2+ or Mg2+.</li>
		<li>Analyze samples immediately or store them in PBS at -80 &#176;C before analysis.</li>
	</ol>
	</li>
</ol>
2. Analysis of Macrophages by Whole-Cell Maldi-TOF MS
<ol>
	<li>Preparation of CHCA matrix.
	<ol>
		<li>Add 500 &#956;l of acetonitrile, 250 &#956;l 10% trifluoroacetic acid, and 250 &#956;l of high-performance liquid chromatography (HPLC) water to a vial. Dilute 10 mg of CHCA in this solution to a final concentration of 10 mg/ml. Mix and sonicate for at least 20 min. 
		Note: sonication will improve the saturation of the matrix but this step is not mandatory.</li>
		<li>Centrifuge at 13,000 x g for 5 min. Discard the pellet and keep the supernatant. 
		Note: prepare the matrix solution just before use. A matrix solution that contains crystals does not allow a good ionization of sample molecules, and this may affect the quality of the spectra.</li>
	</ol>
	</li>
	<li>Preparation of MALDI steel target.
	<ol>
		<li>Moisten the polished steel target with hot tap water. Rub with precision wipe paper. Add 70% ethanol and rub. Rinse with water by rubbing. Add 70% ethanol and rub with precision paper.</li>
		<li>Immerse the target in 70% ethanol and sonicate for at least 15 min. Cover the target with 500 &#956;l to 1 ml&#160;of trifluoroacetic acid at 80%. Rub and wipe with precision paper. Rinse with HPLC water without rubbing. Dry the target at room temperature. 
		Note: an improperly cleaned target may affect the quality of the spectra.</li>
	</ol>
	</li>
	<li>Preparation of deposits.
	<ol>
		<li>Place the clean target on a horizontal and level support to obtain uniform deposits.</li>
		<li>Gently thaw MDM samples on ice. Note: rapid and vigorous thawing may alter samples, thus affecting the quality of the spectra. Homogenize MDMs (by pipetting back and forth the cell suspension) before deposition of 1 &#956;l (containing approximately 1 x 105 cells) on the MALDI target. Add 1 &#956;l of the MALDI matrix solution on the sample. Avoid mixing spot with the pipette. Mixing spots with pipettes alters the quality of the spectra. Depose 12-16 samples/assay (technical replicates).</li>
		<li>Evaporate spontaneously at room temperature. Note: evaporation takes place gradually and leads to the formation of matrix/sample crystals. The deposits may be immediately analyzed or stored in the dark for several days before analysis (up to 2 weeks).</li>
		<li>User should control that the cytokines or heat-killed bacteria used in the experiment do not provide peaks within the range of stimulated macrophages&#39; spectra. Here, we checked that the cytokines and heat-killed bacteria alone did not provide any signal within the studied range (0-20 kDa).</li>
	</ol>
	</li>
	<li>Mass spectrometer tuning and data acquisition.
	<ol>
		<li>Insert the steel target containing samples in the mass spectrometer.</li>
		<li>Configure the mass spectrometer and run data acquisition. Use the default configuration for automated acquisition of the data.</li>
		<li>Note: a detailed view of the configuration of flexControl software is given in the appendix/Table 1. This may vary according to mass spectrometer and software used.</li>
	</ol>
	</li>
</ol>
3. Bioinformatic Analysis
Note: the bioinformatic analysis was performed using the free and open source statistical analysis software R, along with specific analysis libraries (MALDIquant). R can be downloaded freely from its website <a href="http://cran.r-project.org/">http://cran.r-project.org/</a>. A detailed description of the script is provided as supplementary material.
<ol>
	<li>Loading and pretreatment of raw data.
	<ol>
		<li>Store raw data on the computer associated with the mass spectrometer in multiple files and folders. Retrieve the root folder of the experiment with all subfolders. Copy root folder to personal computer for analysis.</li>
		<li>Use readBrukerFlexData and MALDIquant libraries to load and analyze raw data. 
		Note: readBrukerFlexData allows the loading of raw data from the mass spectrometer into a specific object in R for further analysis. See MALDIquant description for further information27.</li>
		<li>Analyze generated spectra. 
		Note: each spectrum consists of a list of peaks with their respective masses and intensities (relative abundance). Use the square root of the intensities to enhance the graphical visualization of the spectra. Correct the background using a statistic-sensitive nonlinear peak-clipping algorithm for baseline estimation27. Use a signal-to-noise ratio of 6 to detect peaks. Consider that the detected peaks are similar across spectra when the mass/charge (m/z) values are within a 2,000 ppm window.</li>
	</ol>
	</li>
	<li>Score definition and computation. Classify the spectra using a hierarchical clustering with a ward algorithm for agglomeration and a dissimilarity matrix based on the Jaccard distance. 
	Note: the Jaccard index measures the similarity between Boolean sample sets (i.e. the presence/absence of a list of peaks).</li>
	<li>Comparison of spectra and viewing.
	<ol>
		<li>Analyze raw spectra plots. Note: x-axis represents the m/z ratio (in Daltons) and the y-axis represents the intensity (relative abundance).</li>
		<li>Assess the similarity between spectra by hierarchical clustering. Represent similarity (or divergence) as dendrogram.</li>
		<li>Assess the reproducibility by the mean of virtual gel-view representation. Note: virtual gel-view representation is a modified heatmap plot where relative abundance is color-coded with increasing intensities of blue.</li>
	</ol>
	</li>
</ol>

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The authors have no conflict of interest to declare. RO, JLM and CC are inventors of an international patent WO 2011/154650, named "Proc&#233;d&#233; d&#39;identification de cellules de mammif&#232;res par spectrom&#233;trie de masse MALDI-TOF".

This protocol describes the use of MALDI-TOF-MS on eukaryotic whole cells. Here, we illustrate the accuracy of the method by analyzing the multiple activation states of macrophages in response to their microenvironment.
The success of the protocol relies on few critical steps. First, any solution contaminant may alter the spectra. For example, it is important to wash cells in PBS to remove culture medium and serum proteins before deposition on the target. A cell concentration of 1 x 105 cells/&#181;l is also needed to ensure reproducible results. Second, the crystallization is an important step in the protocol. To ensure good quality results, the target plate must be carefully washed and the matrix should be prepared before the deposition of samples on the target. The best results are obtained when the samples are deposited on the target just before the matrix solution (avoid mixing the samples with the matrix before the deposition on the target). Correct spontaneous mixing between samples and the matrix solution needs homogeneous deposits. Third, whole-cell MALDI-TOF-MS is a high-throughput technique, which can rapidly result in high amounts of raw data. Bioinformatics analysis is thus a major tool to systematically analyze the data in a reasonable amount of time. Quality assessment, background correction and normalization can be automated. The selection of relevant peaks (e.g. above a given signal-to-noise ratio) and the comparison of spectra based on the presence/absence of these peaks require important computational steps. These methods are described in details in the supplementary material of this article.
Although the acquisition of a mass spectrometer may represent a significant investment, daily running costs are low, and a high number of biological and technical replicates may be easily obtained in one run. For example, our university hospital is able to routinely identify bacteria in 200 clinical samples each day with a similar whole-cell technique. The cell concentration may be a limit for specific clinical applications such as the analysis of needle biopsies or cells harvested from broncho-alveolar lavages. A recent article described an automated approach of whole-cell MALDI-TOF analysis that allowed the robust classification of samples with as few as 250 cells on each spot11. A proof of concept of the clinical application of this technique to the diagnosis of oral cancer has also been recently published15. The matrix choice may limit the type of analyzed molecules. Some matrices will favor the ionization of specific type of molecules (proteins, lipids, sugars&#8230;) and of a given mass range. In our conditions, we were not able to retrieve good quality spectra with ions above 20 kDa. In our protocol, we focused on the analysis of whole spectra as a fingerprint of a given activation state of cells. Therefore, we did not try to identify the proteins that form the main peaks of the spectrum. The identification of specific biomarkers requires an alternative use of mass spectrometry.
In conclusion, we describe here the application of a whole-cell MALDI-TOF MS approach for the accurate and rapid analysis of macrophage activation. This method allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses to cytokines and bacterial pathogens. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a popular mass spectrometry technique to study biological samples. Using a laser beam and an energy-absorbing matrix allows a soft ionization process: the evaporation and genesis of large mostly single-charged biomolecules. This process is called desorption/ionization, justifying the acronym MALDI. These ions are then accelerated by application of voltage and enter a TOF analyzer that allows the separation of these ions and the quantification of their respective masses1.
MALDI-TOF MS has been extensively used in biology, chemistry, and medicine to identify molecules and biomarkers2-4&#160;or to monitor post-translational modifications on&#160;proteins5,6. Recently, several groups applied MALDI-TOF MS to the identification of microorganisms from clinical samples7,8. This microbiological application is now used routinely in the clinical settings. Whole cell MALDI-TOF has many advantages compared to classical applications of MALDI-TOF MS. Samples containing whole cells are directly processed, avoiding time consuming steps to fractionate or separate large amounts of material. Moreover, no characterization of the various peaks is needed: the whole spectrum is considered as a fingerprint of the sample, and matching algorithms compare the tested spectrum with a database of reference spectra.
We and others have applied this whole-cell analysis technique to eukaryotic cells. Many applications may be derived from this technique: (1) identify the main cell types from a mixed sample9-11; (2) assess the viability of cell cultures over time (including quality control industrial applications)12; (3) monitor activation states of a single cell type13; (4) assess the malignant transformation of a clinical sample14,15.
Here, we describe the use of whole-cell MALDI-TOF MS to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. Macrophages play a pivotal role in the immune response to microbial pathogens. They detect infectious agents in the tissues through pattern recognition receptors able to detect conserved microbial patterns, such as lipopolysaccharide (LPS)16. Macrophages are professional antigen-presenting cells that interact with T cells to mount the adaptive immune response. T cells influence macrophages by releasing cytokines that either reinforce or regulate the microbicidal activity of macrophages. By analogy to the Th1/Th2 lymphocyte polarization, inflammatory, microbicidal, and tumoricidal macrophages have been classified into M1 macrophages and immunoregulator macrophages as M2 macrophages17-19. The term M1 refers to the classical activation of macrophages by type I cytokines, such as interferon (IFN)-&#947; and tumor necrosis factor (TNF), or bacterial products, such as LPS18,20-23, whereas macrophages activated by alternative pathways (interleukin (IL)-4, IL-10, Transforming Growth Factor-&#946;1 are considered M2 macrophages19,24,25. The high phenotypic and functional plasticity of macrophages in response to their microenvironment renders these macrophages useful to analyze subtle changes by a MALDI-TOF MS approach.

whole-cell maldi-tof mass spectrometry is an accurate and rapid method to analyze different modes of macrophage activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

The aim of the present protocol is to demonstrate the accuracy of whole-cell MALDI-TOF MS to assess the responsiveness of macrophages to their microenvironment.
Figure 1 describe preparation of stimulated macrophages from blood samples. Figure 2 represents the analysis of monocytes and MDMs by flow cytometry. Note that monocytes expressed CD14 but not CD68 (Figure 1A). Conversely, MDMs expressed CD68 but not CD14 (Figure 1B).
Figure 3 describes the principle of whole-cell MALDI-TOF MS. Cells are deposited with matrix on the target plate. Within the mass spectrometer, a laser beam induces the desorption and ionization of molecules by shooting multiple times on the sample (240 shots). The produced ions are accelerated by a magnetic field and separated according to their m/z ratio in the tube. The TOF analyzer records the impact of the various ions at the end of the tube. According to the time of flight, each impact is converted into a m/z ratio, and impacts corresponding to the same m/z ratio are summed up to generate the full raw spectrums.
Figure 4 illustrates the role of sample preparations in the interpretation of MALDI-TOF MS results. A good quality spectrum is represented in Figure 4A. It usually contains a major peak around 5 kD (m/z = 4,965). A minimum cell concentration is required to obtain good samples: Figure 4B shows a poor quality spectrum obtained with a low cell concentration. However, raising MDM concentration above 1 x 105/&#181;l&#160;does not improve the quality of spectra. Similar poor results are obtained when the sample is mixed with matrix before deposition on the target plate. If mixing is done on the target plate, it may also result in heterogeneous crystallization, as shown in Figure 4C. Hence, deposition of the samples on the target is a tricky and critical step in this protocol.
The reproducibility of the spectra is shown in Figure 5. Here, spectra from various samples are represented as a heatmap. Relative abundance (intensity) is color-coded by intensities of blue. This virtual gel-view representation illustrates the reproducibility of the samples within each class. The normalization and alignment of the spectra is a critical step to obtain such results. An unsupervised analysis by hierarchical clustering is summarized as a dendrogram on the right hand side of the figure. It illustrates that all samples clustered within three different groups: unstimulated MDMs (NS), IFN-&#947;-stimulated or IL-4-stimulated MDMs.
Figure 6 illustrates the discrimination of M1 macrophages (MDMs stimulated with IFN-&#947;) from M2 macrophages (MDMs stimulated with IL-4) and unstimulated MDMs. Indeed, the peak representation of a reference spectrum for IFN-&#947;, IL-4 or unstimulated MDMs shows specific peaks for each class. This representation is obtained using the R MALDIquant library27.
Figure 7 illustrates the specific fingerprints induced by several agonists. It is commonly accepted that IFN-&#947;, TNF and LPS induce an inflammatory (M1-type) response in macrophages. We used MDM samples stimulated with these cytokines alone or in combination to illustrate the accuracy of whole-cell MALDI-TOF MS. Indeed, spectra from all stimulated samples were clearly separated from those of unstimulated macrophages (Figure 7A). However, we obtained a specific fingerprint from each type of stimulation, as illustrated by the clustering of the samples according to the stimuli. Interestingly, MDMs also exhibited specific fingerprints induced by heat-killed bacteria (Figure 7B). These results support the hypothesis that MALDI-TOF MS may be used to analyze circulating cells to assess the host-response to infection or inflammatory diseases in the clinical setting.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig1highres.jpg" src="/files/ftp_upload/50926/50926fig1.jpg" /> 
Figure 1. Preparation of biological samples. 1. Monocytes were selected from peripheral blood mononuclear cells (PBMC) by positive selection with magnetic beads coated with anti-CD14+ antibodies. 2. Macrophages were obtained in 7 days by culture of monocytes in RPMI. 3. Stimulated samples were obtained by adding either cytokines or heat-killed bacteria for 18h on differentiated macrophages. RBC: Red blood cells.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50926/50926fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig2highres.jpg" src="/files/ftp_upload/50926/50926fig2.jpg" /> 
Figure 2. Assessment of CD14 and CD68 expression by flow cytometry. Monocytes (left panel) or MDMs (right panel) were labeled with anti-CD14-PE and anti-CD68-AF647 antibodies to assess membrane expression of these molecules. The differentiation of monocytes into MDMs is accompanied by the down-modulation of CD14 expression and the up-modulation of CD68 expression.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50926/50926fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig3highres.jpg" src="/files/ftp_upload/50926/50926fig3.jpg" /> 
Figure 3. Principle of MALDI-TOF MS technology. This drawing describes the principle of the MALDI-TOF mass spectrometry.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50926/50926fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig4highres.jpg" src="/files/ftp_upload/50926/50926fig4.jpg" /> 
Figure 4. Whole-cell MALDI-TOF MS spectra. A zoomed view of spots deposited on the MALDI target (left panels) with corresponding spectra (right panels). Note that a good quality spot leads to an accurate spectra (A) whereas bad quality spots lead to spectra with a very poor signal to noise ratio (B, C).
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50926/50926fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig5highres.jpg" src="/files/ftp_upload/50926/50926fig5.jpg" /> 
Figure 5. Reproducibility of MALDI-TOF MS spectra. Virtual gel-view of the whole spectra obtained from control and IL-4- or IFN-&#947;-stimulated MDMs are presented as a heatmap. Horizontal axis refers to the m/z ratio. Spectra are classified according to the presence/absence of peaks. NS: non stimulated; IFN-&#947;: interferon-gamma; IL-4: interleukin 4. This figure was reproduced from Ouedraogo&#160;et al.13&#160;with permission.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50926/50926fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig6highres.jpg" src="/files/ftp_upload/50926/50926fig6.jpg" /> 
Figure 6. Reference spectra for M1 and M2 macrophages. The reference spectra for IFN-&#947;- and IL-4-stimulated MDMs are compared to the reference spectrum of nonstimulated (NS) MDMs. The peaks that are shared by stimulated and NS MDMs are in black. The peaks that are induced by stimulation are in red, whereas peaks that are detected only in NS MDMs are in green. m/z: mass/charge ratio; IFN-&#947;: interferon-gamma; IL-4: interleukin 4. This figure was reproduced from&#160;Ouedraogo&#160;et al.13&#160;with permission.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50926/50926fig6highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/50926/50926fig7highres.jpg" src="/files/ftp_upload/50926/50926fig7.jpg" /> 
Figure 7. Hierarchical clustering of activated MDMs. MDMs were stimulated with different agonists for 18 hr. The results are shown as hierarchical clustering of the data. MDMs were activated with M1-related agonists (A) and intracellular bacteria or IL-4 (B). Unstimulated MDMs are presented in grey. IFN-&#947;: interferon-gamma; LPS: lipopolysaccharide from Escherichia coli; TNF: tumor necrosis factor, IL-4: interleukin 4; BCG; bacillus Calmette-Gu&#233;rin; C. burnetii: Coxiella burnetii; O. tsutsugamushi: Orientia tsutsugamushi. This figure was reproduced from Ouedraogo&#160;et al.13&#160;with permission.
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Watch this Scientific Journal Video about Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation at JoVE.com

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

whole-cell-maldi-tof-mass-spectrometry-is-an-accurate-rapid-method-to

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Aix Marseille Université. This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

Video: Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

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.

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. ...

Biochemistry

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

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-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;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 na&#239;ve 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.

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.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Assessing Immunomodulatory Effects of Implant Surfaces on Macrophages

Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-&#947; and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked&#160;Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-&#945;) cytokine and CCR7 surface marker compared to M2&#160;macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-&#593; level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.

Here, we present a detailed protocol for assessing the immunomodulatory potential of implant surfaces in vitro, aiming to improve the reliability and reproducibility of current protocols and promoting further research. Secretory cytokine profiles, mRNA expression, and cell surface markers were monitored using blood monocyte-derived macrophages to investigate macrophage polarization cultivated on titanium.

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the ...

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s initiation and resolution phases, significantly affecting protective immunity and immune-mediated pathological injury. While tissue-resident macrophages are key players in maintaining homeostasis in a steady state, large amounts of monocytes are recruited from the peripheral blood into the tissue following damage or inflammatory insults. Monocyte-derived macrophages (M-DM) can differentiate into many dynamic subtypes, and their phenotypes and functions depend on the local tissue environment. 
To compare different stimuli or environmental conditions during M-DM differentiation and polarization, we standardized an in-vitro model of human nonpolarized M-DM M0 and some cardinal cytokine-polarized macrophages, IFN&#947;/LPS-derived M1, IL-4-derived M2a, and IL-10 or dexamethasone-derived M2c, to analyze skewing reprogramming of M-DM by flow cytometry and real-time PCR. We found that CD64, CD206, CD163, CD14, and MERTK can clearly discriminate unpolarized M0 and polarized M1, M2a, and M2c by flow cytometry. Moreover, we defined IRF1 and CXCL10 as specific genes for classical IFN&#947;/LPS-derived M1-, IRF4-, CCL22-, and TGM2-specific transcripts for IL-4-derived M2a, and the MERTK gene for dexamethasone-derived M2c. To summarize, our standardized M-DM protocol could give the cardinal in-vitro map to analyze the differentiation and polarization of human M-DM under diverse stimuli.

Monocytes and macrophages are very plastic and reprogrammable immune cells crucial for health and disease. They sense and respond to a broad range of stimuli by adopting specific differentiation programs and phenotypes. We have standardized an in-vitro model to study macrophage polarization and reprogramming, providing a valuable tool for research.

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

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Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Proteomic Profiling of Macrophages by 2D Electrophoresis

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays