This work is funded by the Young Investigator Research Program, from the Centre for Infectious Disease Research (ZINF) from the University of W&uuml;rzburg. Juan C Garcia-Betancur is a PhD fellow from the Graduate School of Life Sciences (GSLS) of the University of W&uuml;rzburg.

1. Labeling B. subtilis and Biofilm Formation Assay
<ol>
 <li>Amplify by PCR the promoter region of the gene of interest. We show as example the cloning of PtapA, the promoter of the genes responsible for the production of TasA matrix protein 26. Clone PtapA into pkm008 vector (created by the Rudner lab, Harvard Medical School. Boston, USA) (Fig. 2).</li>
 <li>Linearize the plasmids by enzymatic digestion (Enzyme recommended, XhoI).</li>
 <li>Induce natural competence in B. subtilis strain 168 by following the one-step protocol previously described by Harwood and Cutting 27.</li>
 <li>Add the linearized plasmids into the culture of competent cells and select for spectinomycin resistance after two hours of incubation.</li>
 <li>Strains obtained have inserted the construct into the neutral amyE locus of B. subtilis by double recombination (Fig 4). To create a double-labeled strain, integrate the second reporter into the neutral locus lacA by using the plasmid pDR183 we present in the figure 3. Insert this reporter using the same technique we described above for the insertion of reporters cloned in pKM008.</li>
 <li>Transfer the reporter from the strain 168 to NCIB3610 that is able to form biofilms. Use the SPP1 phage transduction protocol 28,29. Grow donor strain in TY medium (LB+10mM MgSO4+10&mu;M MnSO4). Mix 200 &mu;l of the culture with 100 &mu;l of phage stock dilution. Add 3 ml of soft agar after 30 min of incubation and allow phage halos to arise at 37 &deg;C.</li>
 <li>Collect the soft agar. Centrifuge it and pass the supernatant thought a syringe 0.22 &mu;m filter. Use this supernatant to infect a culture of the recipient strain grown in TY medium. Add 30 &mu;l to 10 ml of culture diluted 1:10. Incubate for 30 min and select for antibiotic resistance after 24 h of incubation.</li>
 <li>Select a colony and grow it overnight on LB at 37 &deg;C.</li>
 <li>Spot 3 &mu;L of the overnight culture on solid biofilm-inducing medium MSgg 1.5 % agar 30. Allow cells to grow during 72 hours at 30 &deg;C (Figure 3). After three days of growth, biofilms formed on the surface of the MSgg agar developed a complex morphological architecture in the surface of the agar.</li>
</ol>
2. Biofilm Dispersion and Cell Fixation
<ol>
 <li>Remove the biofilm form the surface of the MSgg agar using a toothpick or tweezers. The consistency of the biofilm should allow you to to peel it off from the surface of the agar in one piece.</li>
 <li>Place the biofilm in 3 ml of PBS buffer and disperse the biofilm by repetitive passage through a pipette or a needle. Alternatively, biofilm dispersion can be done using mild sonication. Mild sonication requires 12 pulses with an output of 3 and amplitude of 0.7 seconds.</li>
 <li>Fix samples prior cell-single analysis. Resuspend 300 &mu;L of the cell suspension in 1 ml of 4% paraformaldehyde solution and incubate at room temperature for exactly seven minutes. 
 Composition of 4% paraformaldehyde solution: 
 2 g of paraformaldehyde 
 50 ml of PBS buffer 
 4 &mu;l 10 N NaOH 
 Filter the solution through a 0.22 um filter and aliquot</li>
 <li>Wash cells after fixation in PBS buffer three times and resuspend them in 300 &mu;L of PBS buffer.</li>
</ol>
3. Fluorescence Microscopy
<ol>
 <li>Pour 200 &mu;L of 0.8% agarose over a microscope slide and carefully cover it with another slide. Remove the upper slide softly after 2 minutes to obtain a layer of agarose attached to the slide of the bottom.</li>
 <li>Spot 2 &mu;L of fixed cells on the surface of the agarose layer and cover it with a microscope cover glass.</li>
 <li>Place the sample in the fluorescence microscope. We use a Fluorescence microscope Leica DMI6000B equipped with a Leica CRT6000 iIlumination system. The filters for YFP are Ex: BP500/20, Em: BP535/30 and for CFP are Ex: BP426/20, Em: 480/40</li>
 <li>Expose your sample to an excitation fluorescence between 50-200 ms. Set the excitation period according to a negative control which shows no fluorescence in the conditions selected for the experiment.</li>
 <li>Refer the fluorescence image to the same image obtained with bright field. Merge the two images in one picture. Results obtained from flow fluorescence microscopy using a single-labeled strain harboring the reporter PtapA-YFP are represented in figure 6.</li>
</ol>
4. Quantification of Single Cells Using Flow Cytometry
<ol>
 <li>Disperse the sample of fixed cells using mild sonication. Sonicate the sample performing 2 series of 12 pulses with an output of 5 and amplitude of 0.7 seconds, to disperse clumps into single cells without causing cell lysis. Confirm the efficiency of cell dispersion by light microscopy.</li>
 <li>Dilute sample 1:100 in PBS buffer before flow cytometry analysis. We use a flow cytometer BD FACS Canto II. For YFP fluorescence, use a laser excitation at 488 nm coupled with a 530/30 filter. For CFP fluorescence, use the laser excitation at 405 nm coupled with a 408/40 filter.</li>
 <li>Calibrate the flow cytometer machine with two negative controls. A sample of PBS buffer with no cells in suspension should serve as negative control for the size of the particles sensed by the flow cytometer. A sample labeled with no fluorescence reporter should be serve as negative control for the fluorescence sensitivity of the flow cytometer.</li>
 <li>Place the sample labeled with the fluorescent reporter in the flow cytometer. For each sample, analyze at least 50,000 events with a flow rate between 300 and 3000 events per second.</li>
 <li>Capture data using FACS Diva software (BD Biosciences) and analyze it using FlowJo 8.5.2 software. Refer fluorescence signals to the control that shows no fluorescence.</li>
 <li>Present the data monitoring the fluorescence signal of a single-labeled strain in a two-axes graphic. Plot the fluorescence signal detected in the X axis and the number of cells expressing the different levels of fluorescence in the Y axis. Results obtained from flow cytometry analysis using a single-labeled strain harboring the reporter PtapA-YFP are represented in figure 8.</li>
 <li>Present the data monitoring the fluorescence signal of a double-labeled strain in a three-axes graphic. Plot the fluorescence signal of each one of the channels monitored in X and Y axes (for example, GFP would be measured in the X-axis and CFP in the Y-axis). Plot in the Z-axis the number of cells expressing each reporter and present them as contour isolines that would be perpendicular to the plane of the paper (Fig. 9 and 10).</li>
</ol>
4. Representative Results
When B. subtilis grows on a plate of the biofilm-inducing medium MSgg, biofilm formation is observed after three days of incubation at 30 &deg;C 30. The biofilm shows strong consistency and it could be peeled off from the surface of the agar in one piece. Moreover, the biofilm shows a complex morphological architecture that is indicative of the distinct participating cell subpopulations (Fig. 5). For instance, production of the extracellular matrix in biofilms results in the formation of wrinkles on the surface of the colony. This feature can be correlated with the differentiation of the subpopulation of matrix producers 19. Similarly, the raising of aerial structures on the surface of the biofilm is indicative of the presence of a subpopulation of sporulating cells, since the spores localized in the apical area of these structures 30.
Examples of visualization of cell differentiation in a single-labeled and a double-labeled strain using fluorescence microscopy are represented in figure 6 and 7, respectively. The single-labeled strain harbors the fluorescent reporter PtapA-CFP that is expressed in the subpopulation of matrix producing cells. This subpopulation is responsible to produce and secrete the extracellular matrix that constitutes the biofilm (Fig. 6). The double-labeled strain harbors the reporter PtapA-CFP 26 and the additional reporter PsrfAA-YFP 31. This second reporter allows to monitor the subpopulation of cells responsible to secrete the signaling molecule surfactin, which triggers the signaling cascade to the differentiation of the matrix producers (Fig. 7). Subpopulation of matrix producers are false colored in blue while the subpopulation of surfactin producers is false colored in yellow.
Flow cytometry analysis using a single-labeled strain harboring the reporter PtapA-YFP is presented in figure 8. Untreated control strain not harboring any fluorescent protein genes showed a single population low relative fluorescence. Cells harboring PtapA-YFP in non biofilm-inducing conditions did not differentiate the subpopulation of matrix producers and the whole population showed low relative fluorescence. In biofilm-inducing condition, a subpopulation of cells with high relative fluorescence occurred, observed as a shoulder to the right of the low relative fluorescence peak 23.
Results obtained from flow cytometry analysis using a double-labeled strain are represented in figure 9 and 10. Figure 9 monitored the subpopulations of matrix producers and surfactin producers using the double-labeled strain PtapA-CFP, PsrfAA-YFP. As control of background fluorescence we used a strain not harboring any fluorescent protein genes. Next, we detected each subpopulation of matrix producers and surfactin producers in each fluorescence channel, using single-labeled strains as controls. The double-labeled strain PtapA-CFP, PsrfAA-YFP showed two subpopulations of cells expressing high levels of the fluorescent reporters. Each population is framed, showing that there is no overlap in the expression of the reporters between the two subpopulations of specialized cells 15. Similarly, flow cytometry analysis using double-labeled strain Pskf-YFP, PtapA-YFP is presented in figure 10. The reporter for the gene skf monitors the differentiation of the subpopulation of cannibals 32, which has been described to differentiate coordinately with the subpopulation of matrix producers 24. In this case, the double-labeled strain showed a single subpopulation of fluorescent cells expressing both YFP and CFP. This indicated that both cell differentiation pathways are coordinately activated in the same subpopulation.
<img alt="Figure 1" src="/files/ftp_upload/3796/3796fig1.jpg" /> 
Figure 1. Overall scheme of the Experiment. The protocol is divided in three main steps. The first step requires labeling the strain of B. subtilis with the reporter fusion that monitors the subpopulation of interest. Second, grow labeled strains in biofilm-inducing conditions. Third, disperse the biofilm and carry out single-cell analysis of the population using fluorescence microscope and flow cytometry.
<img alt="Figure 2" src="/files/ftp_upload/3796/3796fig2.jpg" /> 
Figure 2. Schematic representation of the integration vector pKM008. This vector integrates the reporter fusion of interest into the neutral locus amyE by double recombination. The promoter of interest (PtapA) is cloned into vector using the restriction sites EcoRI and HindIII. Then, the expression of the cfp gene is under the control of the promoter PtapA. The orientation of the genes in the plasmid is represented as an arrow.
<img alt="Figure 3" src="/files/ftp_upload/3796/3796fig3.jpg" /> 
 Figure 3. Schematic representation of the integration vector pDR183. This vector integrates the reporter fusion of interest into the neutral locus lacA by double recombination. The fusion of interest (PtapA-cfp) is cloned into vector using the restriction sites EcoRI and BamHI. The orientation of the genes in the plasmid is represented as an arrow.
<img alt="Figure 4" src="/files/ftp_upload/3796/3796fig4.jpg" /> 
Figure 4. Scheme of the integration of the reporters into the chromosome of B. subtilis by double recombination. (A) B. subtilis chromosome has two neutral loci, amyE and lacA, to integrate reporter fusions without affecting the development of the biofilm. (B) Process of double recombination of pKM008. Linearized plasmid integrates into the genome of B. subtilis by double recombination. The reporter fusion integrates into the neutral locus in a stable manner.
<img alt="Figure 5" src="/files/ftp_upload/3796/3796fig5.jpg" /> 
Figure 5. Biofilm formation of B. subtilis NCIB3610. Process of bioflm formation of the strain B. subtilis NCIB 3610 when growing on the biofilm-inducing medium MSgg during three days at 30 &deg;C. Sequential pictures of the development of biofilm were taken every 12h.
<img alt="Figure 6" src="/files/ftp_upload/3796/3796fig6.jpg" /> 
Figure 6. Visualization of the subpopulation of matrix producers under the fluorescence microscope. A sample from a biofilm of B. subtilis PtapA-cfp was fixed and examined under the fluorescence microscope. 200 ms of fluorescence excitation evidenced a subpopulation of cells emitting higher fluorescence than the rest of the cells. We considered this subpopulation as the subpopulation of matrix-producing cells. Scale bar is 3 &mu;m.
<img alt="Figure 7" src="/files/ftp_upload/3796/3796fig7.jpg" /> 
Figure 7. Visualization of the subpopulation of matrix producers and surfactin producers under the fluorescence microscope. Sample from a biofilm of the B. subtilis P tapA-cfp, PsrfAA-yfp double-labeled strain was fixed and examined under the fluorescence microscope. Exposure time of 250 ms evidenced two subpopulations of cells emitting higher fluorescence than the rest of the cells. One subpopulation expressed YFP and it was detected exclusively using the YFP channel (false colored in yellow). This is the subpopulation of surfactin producers. Another subpopulation expressed CFP and it was exclusively detected using the CFP channel. This is the subpopulation of matrix producers. Scale bar is 3 &mu;m.
<img alt="Figure 8" src="/files/ftp_upload/3796/3796fig8.jpg" /> 
Figure 8. Quantification of the subpopulation of matrix producers using 2-D flow cytometry. Dispersed cells from a biofilm of B. subtilis PtapA-yfp were monitored using flow cytometry. The flow cytometer counted 50.000 events and the fluorescence signal for each event was monitored. Number of cells counted is plotted in Y axis while the intensity of YFP signal is plotted in the X axis. Cells were grown in LB medium to obtain non-biofilm inducing conditions. Cells were grown in MSgg medium to obtain biofilm-inducing conditions. The subpopulation of matrix producers differentiates only in biofilm-inducing conditions. This figure was adapted from L&oacute;pez et al., PNAS (2009) 106:280-285.
<img alt="Figure 9" src="/files/ftp_upload/3796/3796fig9.jpg" /> 
Figure 9. Quantification of the subpopulation of matrix producers and surfactin producers using 3-D flow cytometry. Fluorescence signal of the channels monitored are presented in the X axis (for YFP) and Y axis (for CFP). The Z-axis measures the number of cells expressing each reporter and it is quantified as contour isolines perpendicular to the plane of the paper. The number of events monitored in this experiment was 50.000 events. Left upper panel presents a control of background fluorescence harboring no fluorescent protein genes. Right upper panel detects the subpopulation of surfactin producers in the YFP fluorescence channel (framed in yellow) using a single-labeled strain PsrfAA-YFP. Left bottom panel detects the subpopulation of matrix producers in the CFP fluorescence channel (framed in blue) using a single-labeled strain PtapA-CFP. The double-labeled strain PtapA-CFP, PsrfAA-YFP is monitored in the right bottom panel. It showed two subpopulations that are framed in yellow and blue. This figure was adapted from L&oacute;pez et al., Genes and Development (2009) 23:1631-1638.
<img alt="Figure 10" src="/files/ftp_upload/3796/3796fig10.jpg" /> 
Figure 10. Quantification of the subpopulation of matrix producers and cannibals using 3-D flow cytometry. Fluorescence signal of the channels monitored are presented in the X axis (for YFP) and Y axis (for CFP). The number of events monitored in this experiment was 50.000. Right upper panel detects the subpopulation of cannibals in the YFP fluorescence channel (framed in yellow) in a single-labeled strain Pskf-YFP. Left bottom panel detects the subpopulation of matrix producers in the CFP fluorescence channel (framed in blue) in a single-labeled strain PtapA-CFP. The double-labeled strain PtapA-CFP, Pskf-YFP showed only one subpopulation of cells in diagonal to the X and Y axes (framed in green). This subpopulation is detected in the YFP and CFP channel because it expresses the two reporters simultaneously. L&oacute;pez et al., Genes and Development (2009) 23:1631-1638.

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E., Ben-Yehuda, S., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruiting+body+formation+by+Bacillus+subtilis.">Fruiting body formation by Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11626 (2001).</li><li>Nakano, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=srfA+is+an+operon+required+for+surfactin+production,+competence+development,+and+efficient+sporulation+in+Bacillus+subtilis.">srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis.</a> J. Bacteriol. 173, 1770-1778 (1991).</li><li>Gonzalez-Pastor, J. E., Hobbs, E. C., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibalism+by+sporulating+bacteria.">Cannibalism by sporulating bacteria.</a> Science. 301, 510-513 (2003).</li><li>Aguilar, C., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+Bacillus+subtilis+as+a+multicellular+organism.">Thinking about Bacillus subtilis as a multicellular organism.</a> Curr. Opin. Microbiol. 10, 638-643 (2007).</li><li>Shapiro, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu. Rev. Microbiol. 52, 81-104 (1998).</li></ol>

We have nothing to disclose.

The fact that bacterial communities show subpopulations of cells expressing specific set of genes evidences the complexity of microbial communities 33,34. This protocol should help to determine whether the expression of any gene of interest is restricted to a particular subpopulation of specialized cells within the microbial community. Visualization of these subpopulations requires the development of new techniques, because traditional methods to monitor gene expression or microarray analysis rate the levels o...

<table border="1">
 <tbody>
 <tr>
 <td>Technique</td>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td rowspan="11">MSgg composition</td>
 <td>potassium phosphate 5mM</td>
 <td>Roth</td>
 <td>6878</td>
 </tr>
 <tr>
 <td>MOPS 100mM</td>
 <td>Sigma-Aldrich</td>
 <td>M1254</td>
 </tr>
 <tr>
 <td>Magnesium chloride 2mM</td>
 <td>Roth</td>
 <td>2189.1</td>
 </tr>
 <tr>
 <td>Calcium chloride 700&mu;M</td>
 <td>Roth</td>
 <td>A119.1</td>
 </tr>
 <tr>
 <td>Ferric chloride 50&mu;M</td>
 <td>Sigma-Aldrich</td>
 <td>157740</td>
 </tr>
 <tr>
 <td>Zinc chloride 1&mu;M</td>
 <td>Applichem</td>
 <td>A2076</td>
 </tr>
 <tr>
 <td>Thiamine 2&mu;M</td>
 <td>Sigma-Aldrich</td>
 <td>74625</td>
 </tr>
 <tr>
 <td>Glycerol 0.5%</td>
 <td>Roth</td>
 <td>7533</td>
 </tr>
 <tr>
 <td>Glutamate 0.5%</td>
 <td>Sigma-Aldrich</td>
 <td>49621</td>
 </tr>
 <tr>
 <td>Tryptophan 50&mu;g/ml</td>
 <td>Sigma-Aldrich</td>
 <td>T0254</td>
 </tr>
 <tr>
 <td>Phenylalanine 50&mu;g/ml</td>
 <td>Sigma-Aldrich</td>
 <td>P2126</td>
 </tr>
 <tr>
 <td>Cell fixation</td>
 <td>Paraformaldehyde</td>
 <td>Roth</td>
 <td>0335</td>
 </tr>
 <tr>
 <td></td>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Sonication</td>
 <td>Cell Sonicator</td>
 <td>Bandelin</td>
 <td>D-1000</td>
 </tr>
 <tr>
 <td>Fluorescence Microscopy</td>
 <td>Fluorescence Microscope</td>
 <td>Leica</td>
 <td>DMI6000B </td>
 </tr>
 <tr>
 <td></td>
 <td>Name of the software</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>Fluorescence Microscopy</td>
 <td>AsaF</td>
 <td>Leica</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow cytometry</td>
 <td>FCASDiva</td>
 <td>BD</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow cytometry</td>
 <td>FlowJo</td>
 <td>Treestar</td>
 <td></td>
 </tr>
 </tbody>
</table>

single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

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Immunology and Infection

20.3K Views.  University of Würzburg. Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Video: Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...