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</...

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).<...

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 ...

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Immunology and Infection

8.7K 조회수.  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.

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

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...