We are thankful to Carlo Manzo and Maria Garc&#237;a Parajo for their help and source code of the diffusion coefficient analysis. This work was supported in part by grants from the Spanish Ministry of Science, Innovation and Universities (SAF 2017-82940-R) and the RETICS Program of the Instituto de salud Carlos III (RD12/0009/009 and RD16/0012/0006; RIER). LMM and JV are supported by the COMFUTURO program of the Fundaci&#243;n General CSIC.

<ol>
	<li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+studies+in+live+cells.">Single-molecule studies in live cells.</a> Annual Review of Pysical Chemistry. 67 (565-585), (2016).</li><li>Mattheyses, A. L., Simon, S. M., Rappoport, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+with+total+internal+reflection+fluorescence+microscopy+for+the+cell+biologist.">Imaging with total internal reflection fluorescence microscopy for the cell biologist.</a> Journal of Cell Science. 123 (Pt 21), 3621-3628 (2010).</li><li>Jaqaman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+single-particle+tracking+in+live-cell+time-lapse+sequences.">Robust single-particle tracking in live-cell time-lapse sequences.</a> Nature Methods. 5 (8), 695-702 (2008).</li><li>Bakker, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+mobility+of+individual+integrin+nanoclusters+orchestrates+the+onset+for+leukocyte+adhesion.">Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion.</a> Proceedings of the National Academy of Sciences U S A. 109 (13), 4869-4874 (2012).</li><li>Kusumi, A., Sako, Y., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confined+lateral+diffusion+of+membrane+receptors+as+studied+by+single+particle+tracking+(nanovid+microscopy).+Effects+of+calcium-induced+differentiation+in+cultured+epithelial+cells.">Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.</a> Biophysical Journal. 65 (5), 2021-2040 (1993).</li><li>Ferrari, R. M., Manfroi, A. J., Young, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongly+and+weakly+self-similar+diffusion.">Strongly and weakly self-similar diffusion.</a> Physica D. 154, 111-137 (2001).</li><li>Sbalzarini, I. F., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feature+point+tracking+and+trajectory+analysis+for+video+imaging+in+cell+biology.">Feature point tracking and trajectory analysis for video imaging in cell biology.</a> Journal of Structural Biology. 151 (2), 182-195 (2005).</li><li>Ewers, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking+of+murine+polyoma+virus-like+particles+on+live+cells+and+artificial+membranes.">Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes.</a> Proceedings of the National Academy of Sciences U S A. 102 (42), 15110-15115 (2005).</li><li>Manzo, C., Garcia-Parajo, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+progress+in+single+particle+tracking:+from+methods+to+biophysical+insights.">A review of progress in single particle tracking: from methods to biophysical insights.</a> Report on Progress in Physics. 78 (12), 124601 (2015).</li><li>Calebiro, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+fluorescently+labeled+G-protein-coupled+receptors+reveals+complexes+with+distinct+dynamics+and+organization.">Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.</a> Proceedings of the National Academy of Sciences U S A. 110 (2), 743-748 (2013).</li><li>Digman, M. A., Dalal, R., Horwitz, A. F., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+number+of+molecules+and+brightness+in+the+laser+scanning+microscope.">Mapping the number of molecules and brightness in the laser scanning microscope.</a> Biophysical Journal. 94 (6), 2320-2332 (2008).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Martinez-Munoz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+Actin-Dependent+Chemokine+Receptor+Nanoclustering+from+Dimerization+Indicates+a+Role+for+Clustering+in+CXCR4+Signaling+and+Function.">Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.</a> Molecular Cell. 70 (1), 106-119 (2018).</li><li>Destainville, N., Salome, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+correction+of+systematic+errors+due+to+detector+time-averaging+in+single-molecule+tracking+experiments.">Quantification and correction of systematic errors due to detector time-averaging in single-molecule tracking experiments.</a> Biophysical Journal. 90 (2), L17-L19 (2006).</li></ol>

The authors have nothing to disclose.

The described method is easy to perform even without having any previous experience working with Matlab. However, Matlab routines require extremely accuracy with the nomenclature of the different commands and the localization of the different folders employed by the program. In the tracking analysis routine (step 3), multiple parameters can be modified. The &#34;Setting Gaussian-Mixture Model Fitting&#34; window (step 3.8) controls how U-track will detect single particles on the video. This is done by fitting a Gaussian ...

Membrane proteins embedded in the lipid bilayer are in continuous movement due to thermal diffusion. Their dynamics are essential to regulate cell responses, as intermolecular interactions allow formation of complexes that vary in size from monomers to oligomers and influence the stability of signaling complexes. Elucidating the mechanisms controlling protein dynamics is thus a new challenge in cell biology, necessary to understand signal transduction pathways and to identify unanticipated cell functions.
Some optical methods have been developed to study these interactions in living cells1. Among these, total internal reflection fluorescence (TIRF) microscopy, developed in the early 1980s, allows the study of molecular interactions at or very near the cell membrane2. To study dynamic parameters of membrane protein trajectories obtained from TIRF data in living cells, a single particle tracking method (SPT)is required. Although several algorithms are available for this, we currently use those published by Jaqaman et al.3 that address particle motion heterogeneity in a dense particle field by linking particles between consecutive frames to connect the resulting track segments into complete trajectories (temporary particle disappearance). The software captures the particle merging and splitting that result from aggregation and dissociation events3. One of the output data of this software is detection of the particles along the entire trajectory by defining their X and Y positions in each frame.
Once particles are detected, we apply different algorithms to determine the short timelag diffusion coefficient (D1-4)4,5. By applying the Moment Scaling Spectrum (MSS)6,7,8 analysis or by fitting the &#39;alpha&#39; value by adjustment of the Mean Square Displacement (MSD) to the curve9, we also classify the particles according to the type of trajectory.
Analysis of spot intensity in fluorescence images is a shared objective for scientists in the field10,11. The most common algorithm used is the so-called Number and Brightness. This method nonetheless does not allow correct frame-by-frame intensity detection in particles in the mobile fraction. We have, thus, generated a new algorithm to evaluate these particle intensities frame-by-frame and to determine their aggregation state. Once the coordinates of each particle are detected using U-Track2 software3, we define its intensity in each frame over the complete trajectory, also taking into account the cell background in each frame. This software offers different possibilities to determine the spot intensity and the cell background and, using known monomeric and dimeric proteins as references, calculates the approximate number of proteins in the particle detected (cluster size).
In this article, we describe a careful guide to perform these three steps: 1) detecting and tracking single particles along a video of fluorescence microscopy using U-track; 2) analyzing the instantaneous diffusion coefficient (D1-4) of those particles and the type of movement (confined, free, or directed) of particles with long trajectories by MSS; 3) measuring the spot intensity along the video corrected by the estimated background fluorescence for each spot. This allows cluster size estimation and identification of the photobleaching steps.
The use of this protocol does not require specialized skills and can be performed in any laboratory with cell culture, flow cytometry and microscopy facilities. The protocol uses ImageJ or Fiji (a distribution of ImageJ12), U-track3, and some ad hoc made routines (http://i2pc.es/coss/Programs/protocolScripts.zip). U-track and ad hoc routines run over Matlab that can be installed in any compatible computer.

<table border="0" cellpadding="0" cellspacing="0" style="width:797px;" width="797">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Human Jurkat cells</td>
			<td style="width:203px;">ATCC</td>
			<td style="width:203px;">CRL-10915</td>
			<td style="width:171px;">Human T cell line. Any other cell type can be analyzed with this software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">pAcGFPm-N1 (PT3719-5)DNA3GFP</td>
			<td style="width:203px;">Clontech</td>
			<td style="width:203px;">632469</td>
			<td>Different fluorescent proteins can be followed and analyzed with this routine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Gene Pulse X Cell electroporator&#160;</td>
			<td>BioRad&#160;</td>
			<td style="width:203px;"></td>
			<td>We use 280V, 975 mF, for Jurkat cells.&#160; Use the transfection method best working in your hands.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytomics FC 500 flow cytometer&#160;</td>
			<td>Beckman Coulter</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MoFlo Astrios Cell Sorter&#160;</td>
			<td>Beckman Coulter</td>
			<td style="width:203px;"></td>
			<td>Depending on the level of transfection, cell sorting may not be required.&#160; You can also employ cells with stable expression of adequate levels of the receptor of interest.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dako Qifikit</td>
			<td style="width:203px;">DakoCytomation</td>
			<td style="width:203px;">K0078</td>
			<td>Used for quantification the number of receptors in the cell surface.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Glass bottom microwell dishes</td>
			<td style="width:203px;">MatTek corporation</td>
			<td style="width:203px;">P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Human Fibronectin from plasma</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:203px;">F0895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Recombinant human CXCL12</td>
			<td style="width:203px;">PeproTech</td>
			<td style="width:203px;">300928A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Inverted Leica AM TIRF</td>
			<td style="width:203px;">Leica</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">EM-CCD camera</td>
			<td style="width:203px;">Andor&#160;</td>
			<td style="width:203px;">DU 885-CSO-#10-VP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">MATLAB</td>
			<td style="width:203px;">The MathWorks, Natick, MA</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">U-Track2 software</td>
			<td style="width:203px;">Danuser Laboratory</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">ImageJ</td>
			<td style="width:203px;">NIH</td>
			<td style="width:203px;"></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">FiJi</td>
			<td style="width:203px;">FiJI</td>
			<td style="width:203px;"></td>
			<td>https://imagej.net/Fiji)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">u-Track2 software</td>
			<td style="width:203px;"></td>
			<td style="width:203px;"></td>
			<td>Matlab tool.&#160; For installing, download .zip file from the web page (http://lccb.hms.harvard.edu/software.html) and uncompress the file in a directory of your choice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">GraphPad Prism</td>
			<td style="width:203px;">GraphPad software</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

image processing protocol for the analysis of the diffusion and cluster size of membrane receptors by fluorescence microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

The use of this protocol allows the automated tracking of particles detected in fluorescence microscopy movies and the analysis of their dynamic characteristics. Initially, cells are transfected with the fluorescently-coupled protein to be tracked. The appropriate level of receptors presents on the cell surface that allows SPT is obtained by cell sorting (Figure 1). Selected cells are analyzed by TIRF microscopy that generates videos in a format that can be s...

Watch this Scientific Journal Video about Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy at JoVE.com

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...

image-processing-protocol-for-analysis-diffusion-cluster-size

Research

JoVE Journal

Immunology and Infection

8.6K Views.  Campus Univ. Autónoma de Madrid. Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Video: Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

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.

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.

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 ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...