The authors would like to thank Dr. Olivia Steele-Mortimer for sharing pCHAR-Duo plasmid. This work was funded by the Italian Ministry of Health, grants PRC2019014 and PRC2021004.

<ol><li>European Food Safety Authority & European Centre for Disease Prevention and Control. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+European+Union+One+Health+2020+Zoonoses+Report%5BTitle%5D)+AND+%22EFSA+Journal%22%5BJournal%5D)">The European Union One Health 2020 Zoonoses Report.</a> EFSA Journal. 19 (12), 6971(2021).</li>
<li>Fattinger, S. A., Sellin, M. E., Hardt, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salmonella+effector+driven+invasion+of+the+gut+epithelium%3A+breaking+in+and+setting+the+house+on+fire%5BTitle%5D)+AND+%22Current+Opinion+in+Microbiology%22%5BJournal%5D)">Salmonella effector driven invasion of the gut epithelium: breaking in and setting the house on fire.</a> Current Opinion in Microbiology. 64, 9-18 (2021).</li>
<li>Knodler, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissemination+of+invasive+Salmonella+via+bacterial-induced+extrusion+of+mucosal+epithelia%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (41), 17733-17738 (2010).</li>
<li>Fulde, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salmonella+SPI2+T3SS+mediates+transcytosis+in+polarized+enterocytes+in+vivo%5BTitle%5D)+AND+%22Cell+Host+%26+Microbe%22%5BJournal%5D)">Salmonella SPI2 T3SS mediates transcytosis in polarized enterocytes in vivo.</a> Cell Host & Microbe. , (2019).</li>
<li>Cooper, K. G., Chong, A., Starr, T., Finn, C. E., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictable%2C+tunable+protein+production+in+Salmonella+for+studying+host-pathogen+interactions%5BTitle%5D)+AND+%22Frontiers+in+Cellular+and+Infection+Microbiology%22%5BJournal%5D)">Predictable, tunable protein production in Salmonella for studying host-pathogen interactions.</a> Frontiers in Cellular and Infection Microbiology. 7, 475(2017).</li>
<li>Klein, J. A., Grenz, J. R., Slauch, J. M., Knodler, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+activity+of+the+Salmonella+invasion-associated+injectisome+reveals+its+intracellular+role+in+the+cytosolic+population%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Controlled activity of the Salmonella invasion-associated injectisome reveals its intracellular role in the cytosolic population.</a> mBio. 8 (6), 01931(2017).</li>
<li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li>
<li>Ibarra, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+Salmonella+pathogenicity+island+1+under+different+growth+conditions+can+affect+Salmonella-host+cell+interactions+in+vitro%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro.</a> Microbiology. 156 (4), 1120-1133 (2010).</li>
<li>Tambassi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutation+of+hilD+in+a+Salmonella+Derby+lineage+linked+to+swine+adaptation+and+reduced+risk+to+human+health%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health.</a> Scientific Reports. 10 (1), 21539(2020).</li>
<li>Finn, C. E., Chong, A., Cooper, K. G., Starr, T., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+second+wave+of+Salmonella+T3SS1+activity+prolongs+the+lifespan+of+infected+epithelial+cells%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells.</a> PLoS Pathogens. 13 (4), 1006354(2017).</li>
<li>Knodler, L. A., Nair, V., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+cytosolic+Salmonella+in+epithelial+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Quantitative assessment of cytosolic Salmonella in epithelial cells.</a> PLoS One. 9 (1), 84681(2014).</li>
<li>Chong, A., Starr, T., Finn, C. E., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+the+Salmonella+Type+III+Secretion+System+1+in+bacterial+adaptation+to+the+cytosol+of+epithelial+cells%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells.</a> Molecular Microbiology. 112 (4), 1270-1283 (2019).</li>
</ol>

The authors declare that they have no competing interests.

The way Salmonella colonizes intestinal epithelial cells, influences the infection outcome. Upon invasion, the cytosolic hyper-replication induces inflammation of the gut3, whereas vacuolar replication can lead to systemic spread4. Salmonella strains can vary in their ability to invade and replicate inside intestinal epithelial cells9. Indeed, Salmonella is an extremely diverse genus comprising more than 2,500 serovars, which have different abilities to cause disease. In addition, Salmonella is the most frequently reported cause of foodborne outbreaks in the European Union, indicating a large spread in the human population1. Therefore, the availability of reliable, high-throughput methods for the quantification of the intracellular phenotypes of Salmonella is of great importance to evaluate differences in virulence among large numbers of Salmonella strains and ultimately allow an accurate and strain-specific risk assessment of this pathogen.
The protocol described here measures the behavior of Salmonella inside epithelial cells in a fast and automated way. The infection of epithelial cells is performed in 96-well imaging microplates and an automated fluorescence microscope is used for image acquisition, making the protocol suitable for high-throughput applications. This protocol takes advantage of the pCHAR-Duo plasmid, which allows distinguishing vacuolar from cytosolic Salmonellae through the differential expression of two fluorescent reporters5. The intracellular phenotypes of Salmonella are often analyzed by manual scoring, a time-consuming procedure that is unsuitable for the analysis of large numbers of strains and cell cultures per strain and prone to operator&#39;s errors and inter-operator variation. To overcome these limitations, two complementary and automated image analyses were developed, the area analysis and the single-cell analysis. ImageJ, a freely available software, was used to develop the two analyses. In order to accelerate the protocol execution, ImageJ scripts for batch analysis of multiple acquisition files with no operator intervention are provided as supplemental files.
The area analysis was designed to quantify, in a few steps, the overall colonization of epithelial cells (infection ratio) and the&#160;hyper-replication level (hyper-replication ratio) through the measurement of the areas specifically occupied by the nuclei of epithelial cells and by Salmonellae expressing either mCherry or mCherry together with GFP. The area analysis is applied to images acquired at low magnification, where both vacuolar and hyper-replicating Salmonellae are in focus in the same z-plane, and a large number of epithelial cells is displayed per microscopic field, reducing the size and number of acquisition files. The area analysis allows for an automated, fast, and computationally-light analysis of large number of samples, making it suitable for high-throughput assays such as screening experiments.
The single-cell analysis was designed to quantify Salmonella phenotypes inside epithelial cells with single-cell resolution, obtained through cell segmentation and measurement of the cellular area and the percentage of area occupied by vacuolar and cytosolic hyper-replicating Salmonellae. The overall colonization of epithelial cells characterized through the area analysis is here broken down into three quantitative parameters, the percentage of infected cells, the mean vacuolar load, and the hyper-replication rate, allowing to evaluate and quantify the contribution of each phenotype to the overall colonization, therefore,&#160;complementing the results of area analysis. The greater details offered by the single-cell analysis come at the cost of slower image acquisition and analysis. In fact, in order to achieve single-cell resolution, image acquisition is performed at high magnification. This implies that multiple z-planes are needed to observe both vacuolar and cytosolic Salmonellae in focus and that the acquisition of a large number of fields per sample is needed to score a high number of epithelial cells, thus extending acquisition time in comparison with area analysis. Furthermore, the analysis of several large acquisition files is computationally demanding, thus requiring a suitable workstation (a 6-core, 32 GB RAM workstation was used). Therefore, single-cell analysis can be used as a stand-alone in limited throughput conditions or as a second-level method coupled to the area analysis to gain a deeper understanding of the Salmonella phenotypes inside epithelial cells.
The two complementary analyses were validated by using S. Tm, S. Derby wt, and S. Derby &#916;sipA, chosen because their behavior inside epithelial cells was already known to differ in terms of invasion or replication9,10. The results of the area and single-cell analyses show that the protocol allowed to quantitatively distinguish differences in intracellular Salmonella phenotypes in accordance with the characteristics of the tested strains. Furthermore, these results demonstrate that the single-cell analysis allows for quantification of the contribution of each intracellular phenotype (invasion, vacuolar load&#160;and cytosolic replication) to the overall colonization scored using the area analysis.
This protocol was applied here to study in vitro pathogenicity of different strains of Salmonella, but it can have other applications such as the study of random mutants to identify genes involved in the invasion and/or replication inside epithelial cells. In addition, the protocol can be adapted to analyze the behavior of Salmonella inside other cell lines than INT407 cells. It can also be used as the starting point to develop similar methods for studying cell-pathogen interaction of other intracellular microorganisms.

Salmonella is the most frequently reported bacterial agent causing outbreaks of foodborne disease in the European Union1. The primary pathological manifestation of Salmonella infection is enteritis, which is the result of the pathogen behavior in the gut following ingestion and the consequent local inflammatory response2. However, Salmonella can also disseminate to extra-intestinal sites and cause systemic infection, especially in immunocompromised individuals. The type of interaction between Salmonella and the intestinal epithelium conditions the outcome of the infection. Once in the gut lumen, Salmonella invades and replicates inside intestinal epithelial cells. At the intracellular level, Salmonella can present two different replication phenotypes, the cytosolic hyper-replication and the intravacuolar slow replication within Salmonella-containing vacuoles (SCVs). The cytosolic hyper-replication induces inflammatory host cell death and Salmonella extrusion into the gut lumen3; the vacuolar replication leads to a trans-epithelium penetration and systemic spread4. Therefore, the extent of invasion and vacuolar vs. cytosolic replication influences the course of infection.
The genus Salmonella is very diverse, including thousands of serotypes with different host-ranges and abilities to cause disease. For example, S. Typhimurium is defined as a generalist serovar, because it infects multiple unrelated hosts, and represents one of the major causes of human salmonellosis. Differently, S. Derby is considered a swine-adapted serovar, as it is mostly isolated from the swine, but it is also reported in the top five of the serovars responsible for human infection1. However, knowledge about the bacterial behavior inside the epithelial cells is essentially limited to the study of a few reference strains, as S. Typhimurium SL1344, that do not represent the vast natural diversity of Salmonella pathogenicity. Characterizing the interaction of different strains of Salmonella with epithelial cells would contribute to understanding their different pathogenicity. For this reason, a high-throughput fluorescence microscopy-based protocol was developed to analyze the intracellular behavior of a large number of strains in a fast and largely automated way. In this protocol, infection of epithelial cells was performed in 96-well imaging plates and image acquisition was made using an automated fluorescence microscope. The pCHAR-Duo plasmid was used to observe the invasion and replication phenotypes of Salmonella inside epithelial cells through fluorescent microscopy5. This plasmid carries the gene encoding the red fluorescent reporter mCherry, constitutively expressed by all the transformed bacterial cells, and the gene encoding the green fluorescent reporter GFP, whose expression is activated by glucose-6-phosphate present exclusively in the cytosol of eukaryotic cells and absent in SCVs. Therefore, the plasmid allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP reporters.
The vacuolar and cytosolic bacteria on microscopy images are commonly quantified by manual scoring6, but this is a time-consuming method that limits analysis to a small number of samples. Therefore, two complementary and automated image analyses were developed-area analysis and single-cell analysis-using ImageJ7, a freely available image analysis software. The area analysis measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication by using data of areas occupied by epithelial cells, red and green Salmonellae in each acquired microscopy image. This method can be applied to images acquired at low magnification; therefore, it allows to score a high number of epithelial cells with few images, shortening the acquisition time. The single-cell analysis uses cell segmentation to determine the percentage of infected cells, the mean vacuolar load, and the percentage of infected cells undergoing cytosolic hyper-replication with single-cell resolution.
In this protocol, all steps of the image analysis are described in detail to be performed manually, but the same analysis can be automated by our specifically designed ImageJ scripts. These scripts also allow to run batch analyses to automatically analyze multiple images and thus speed up the execution of the method.

<table><tbody><tr><td>96-well imaging microplate</td><td>Eppendorf</td><td>30741030</td><td>Cell culture and infection</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>59349</td><td>Bacteria culture</td></tr><tr><td>Axio observer inverted microscope</td><td>ZEISS</td><td></td><td>Automated fluorescence microscope</td></tr><tr><td>Axiocam 305 Mono</td><td>ZEISS</td><td></td><td>Microscope Camera</td></tr><tr><td>Breathable sealing membrane</td><td>Sigma-Aldrich</td><td>Z380059</td><td>Infection assay</td></tr><tr><td>Colibr&amp;igrave; 5/7</td><td>ZEISS</td><td></td><td>Led light source</td></tr><tr><td>Collagen I rat tail</td><td>Life Technologies</td><td>A1048301</td><td>Collagen coating</td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td>Cell staining</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10099-141</td><td>Cell culture and infection</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G12664</td><td>Infection assay</td></tr><tr><td>Glacial acetic acid</td><td>Carlo Erba</td><td>401391</td><td>Collagen coating</td></tr><tr><td>HCS CellMask Blue</td><td>Invitrogen</td><td>H32720</td><td>Cell staining</td></tr><tr><td>ImageJ</td><td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin)</td><td></td><td>Image processing software</td></tr><tr><td>Minimum Essential Eagle&#039;s Medium</td><td>Sigma-Aldrich</td><td>M5650-500ML</td><td>Cell culture and infection</td></tr><tr><td>Paraformaldehyde 4%</td><td>Invitrogen</td><td>FB002</td><td>Cell fixation</td></tr><tr><td>Potassium chloride</td><td>PanReac AppliChem</td><td>131494.1211</td><td>PBS preparation</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>60220</td><td>PBS preparation</td></tr><tr><td>Sodium Chloride</td><td>PanReac AppliChem</td><td>131659.1214</td><td>Bacteria culture and PBS preparation</td></tr><tr><td>Sodium phosphate Dibasic</td><td>Sigma-Aldrich</td><td>71640</td><td>PBS preparation</td></tr><tr><td>Tissue Culture Flask 25 cm&lt;sup&gt;2&lt;/sup&gt; plug seal screw cap</td><td>Euroclone</td><td>ET7025</td><td>Cell culture</td></tr><tr><td>Triton X-100</td><td>Biorad</td><td>1610407</td><td>Cell staining</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200056</td><td>Cell culture and infection</td></tr><tr><td>Tryptone</td><td>Oxoid</td><td>LP0042B</td><td>Bacteria culture</td></tr><tr><td>Yeast extract</td><td>Biolife</td><td>4122202</td><td>Bacteria culture</td></tr></tbody></table>

automated analysis of intracellular phenotypes of salmonella using imagej

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Infection of epithelial cells with Salmonella strains 
This protocol was developed to analyze the cellular invasion and the cytosolic replication (Figure 1A) vs vacuolar load&#160;(Figure 1B) of Salmonella inside epithelial cells. The protocol was validated by using the three following Salmonella strains, S. Typhimurium SL1344 reference strain (S. Tm), S. Derby ER1175 wildtype (S. Derby wt) and the isogenic mutant of S. Derby ER1175 without sipA gene (S. Derby &#916;sipA). S. Derby ER1175 strain was isolated from swine and belongs to the IZSLER surveillance collection of Salmonella isolates. The strains were selected in order to represent the phenotype diversity covered by the protocol. In particular, these strains were chosen because their behavior inside epithelial cells was already known to differ in terms of invasion or replication9; therefore, they were valuable controls to test whether the protocol allowed to quantitatively distinguish differences in intracellular Salmonella phenotypes. In particular, S. Tm and S. Derby wt were included because we had previously demonstrated that S. Tm has higher invasion and intracellular replication efficiency than S. Derby wt9 while S. Derby &#916;sipA was added as a hyper-replication impaired strain since the virulence effector SipA plays a crucial role in the onset of hyper-replication10,11. It was previously reported for S. Tm that cytosolic replication starts&#160;4 h post-invasion, and then the cytosolic population rapidly hyper-replicates to fill the epithelial cell by 8 h11,12. Consistently, 8 h long infection was suitable to observe and quantify the cytosolic hyper-replication phenotype also in S. Derby wt and S. Derby &#916;sipA.
Area analysis 
The area analysis in step 4.1 provides a measurement of the overall colonization of epithelial cells by Salmonella (infection ratio), together with a measurement of the hyper-replication (hyper-replication ratio). In the area analysis workflow described in Figure 2, the random noise is reduced, and then channels are split and processed independently. A threshold is set for each channel in order to exclude the background from the area measurement. Then, the area occupied by thresholded pixels is measured for each channel. The output of area analysis is a table reporting, for each acquisition file, the extension of the areas occupied by epithelial cell nuclei (blue channel), intracellular mCherry-expressing Salmonellae (red channel), and cytosolic hyper-replicating Salmonellae that express GFP (green channel) along with mCherry. The infection ratio is calculated by dividing the area occupied by mCherry-expressing Salmonellae by the area occupied by host cell nuclei. The results of the tested strains showed that S. Tm displays a significantly higher infection ratio compared to both S. Derby strains, as expected (Figure 3A). Therefore, these results demonstrate the efficacy of area analysis in detecting differences in the ability of Salmonella strains to colonize epithelial cells. Salmonella hyper-replication ratio is measured by dividing the area occupied by GFP-expressing Salmonellae by the area occupied by intracellular mCherry-expressing Salmonellae. Consistently with the role of SipA in determining hyper-replication, a significantly reduced hyper-replication ratio for S. Derby &#916;sipA strain was measured compared to S. Derby wt, (Figure 3B). No hyper-replication difference was observed between S. Tm and S. Derby wt. Overall, area analysis effectively revealed different hyper-replication levels among the assayed strains.
Single-cell analysis 
The single-cell analysis allows quantifying Salmonella invasion and vacuolar load vs. cytosolic replication inside epithelial cells with single-cell resolution. As described in step 4.2, for each acquisition file, blue, red, and green channels were processed independently (Figure 4). Specifically, the blue channel, corresponding to epithelial cells, was processed in step 4.2.3 to obtain cell segmentation. Then, segmented cells were added to the Region of Interest (ROI) Manager to obtain a list of ROIs, corresponding to all segmented cells uniquely labeled with y-x coordinates. In order to remove out-of-focus pixels of GFP- expressing hyper-replicating Salmonellae, the pixels of the green channel were subtracted from those of the red channel. The output table of single cell analysis reports, for each ROI, the area and the percentage of the ROI&#39;s area occupied by Salmonellae expressing the mCherry constitutive reporter only or the GFP cytosol-responsive reporter. The percentage of the cell&#39;s area occupied by mCherry-only expressing Salmonellae was processed in step 4.2.4 to calculate the percentage of infected cells and the mean vacuolar load (Figure 5A).&#160;Analysis of the vacuolar load showed that the S. Derby strains generate a mean vacuolar load significantly lower than S. Tm (Figure 5B). Conversely, only a slight and not significant reduction of the percentage of infected cells was observed in S. Derby strains compared to S. Tm (Figure 5A). The percentage of the cell&#39;s area occupied by GFP-expressing Salmonellae was processed in step 4.2.5 to obtain the hyper-replication rate. No difference was observed between S. Tm and S. Derby wt, while S. Derby &#916;sipA displayed a significant decrease of hyper-replication, consistent with the result of the area analysis (Figure 5C)&#160;and the role of SipA in inducing hyper-replication.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64263/64263fig1v2.jpg" /> 
Figure 1: Salmonella cytosolic hyper-replication and vacuolar load. Representative images of (A) the Salmonella hyper-replication&#160;and (B) the vacuolar load&#160;acquired at high magnification (40x) are shown. Panel B shows the different focus planes of cells containing hyper-replicating Salmonellae (z:7/8), compared to non-hyper-replicating Salmonellae (z:4/8). White scale bars are 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64263/64263fig02.jpg" /> 
Figure 2: Workflow of the area analysis. The workflow of the area analysis is shown for a representative low magnification (10x) acquisition of INT407 cells infected for 8 h with Salmonella carrying the pCHAR-Duo plasmid (MOI 100). First, the random noise is reduced, and then the channels are split into C1, C2, and C3 and processed independently. A threshold is set for each channel in order to exclude the background from the area of measurement. Then, the area occupied by the thresholded pixels only-corresponding to cell nuclei in C1, all intracellular Salmonellae in C2, and cytosolic Salmonellae in C3-is measured for each channel. The images analyzed here are the results of four tiles acquired at 10x magnification fused together by stitching. White scale bars are 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64263/64263fig03.jpg" /> 
Figure 3: Results of the area analysis. The results of the area analysis are shown. (A) Infection ratio is calculated by dividing the area occupied by mCherry-expressing Salmonellae (C2 channel), representing all the intracellular bacteria, by the area occupied by host cell nuclei (C1 channel). (B) Hyper-replication ratio was measured by dividing the area occupied by GFP-expressing Salmonellae (C3 channel), representing cytosolic hyper-replicating bacteria only, by the area occupied by all intracellular mCherry-expressing Salmonellae. Each dot represents a replicate. The analysis was conducted on three biological replicates, each tested in triplicate. The black lines indicate the mean values. The significance was calculated using a two-tailed t-test, and p values are reported. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64263/64263fig04.jpg" /> 
Figure 4: Workflow of the single-cell analysis. The workflow of the single-cell analysis is shown for a representative high magnification acquisition (40x) of INT407 cells infected for 8 h with Salmonella carrying the pCHAR-Duo plasmid (MOI 100). First, channels are split into C1, C2, and C3 and processed independently. Blue channel (C1), corresponding to epithelial cells, is processed to obtain cell segmentation, and then each segmented cell is defined as a Region of Interest (ROI) uniquely labeled with y-x coordinates. The red channel (C2) is processed by subtracting the green channel (C3) to remove out-of-focus cytosolic hyper-replicating Salmonellae, leaving all vacuolar Salmonellae expressing mCherry only, and then random noise was reduced, and the threshold is set to exclude the background from measurements. Finally, the area occupied by vacuolar Salmonellae for each ROI is measured. The green channel (C3), corresponding to total cytosolic GFP-expressing Salmonellae, is processed similarly. The images analyzed here are the results of 16 tiles fused together by stitching. White scale bars are 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64263/64263fig05.jpg" /> 
Figure 5: Results of the single-cell analysis. The results of the single-cell analysis are shown. (A) The percentage of infected cells is obtained by dividing the number of cells with a percentage of area occupied by mCherry-only expressing Salmonellae&#62; 0.2 by the total number of cells. (B) The mean vacuolar load was obtained by calculating the mean percent area occupied by mCherry-only expressing Salmonellae in the infected cells. (C) The hyper-replication rate was calculated by dividing the number of cells with a percentage of area occupied by GFP-expressing Salmonellae&#8805;20% by the total number of infected cells. Data from three to four biological replicates tested in triplicate are reported. The bars indicate the standard error of measurement. The significance was calculated using a two-tailed t-test, and p values are reported. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Area analysis script. <a href="https://www.jove.com/files/ftp_upload/64263/REVIEWED_Area analysis script.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 2: Single-cell analysis script. <a href="https://www.jove.com/files/ftp_upload/64263/REVIEWED_Single-cell analysis script.zip" target="_blank">Please click here to download this File.</a>

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Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

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2.8K Views.  Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (IZSLER). Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Video: Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Bioengineering

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Identifying Drugs that Affect Polymerization of Bacterial Cytoskeletal Proteins

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

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Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins