This project was supported partly by a grant for National Institutes of Health NIAID (for A.J.B. and L.G.A., R01 AI076246). The Salmonella mutant collection was partly supported by National Institutes of Health grants (for M.M., U01 A152237-05, R01 AI07397-01, R01 AI039557-11 and R01 AI075093-01), partly by National Institutes of Health grants (for H.A.P, R21 AI083964-01, 1R0 1AI083646-01, 1R56AI077645, R01 AI075093). We thank Steffen Prowollik for replica plating and confirming the mutants in the collection.

1. Murine Macrophage RAW264.7 Cell Culture
<ol>
	<li>Grow low passage number murine macrophage cells, RAW264.7 (The ATCC&#174; Number, TIB-71) in a T-75 cell culture flask vented filter cap in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.5% NaHCO3, and 1% 100x Nonessential amino acids (NEAA) at 37&#176;C, in a 5% CO2 incubator.</li>
	<li>Once cells reach a 60-80% confluence in the flask, use a cell scraper to harvest cells, and count the cells in a hemacytometer and calculate the cell concentration.</li>
	<li>Resuspend the cells and dilute the cell concentration into 2.5 x 105/ml in fresh DMEM cell culture medium. Use multichannel pipettes to plate 200 &#956;l of DMEM cell culture medium containing 5 x 104 cells in each well of 96-well cell culture plates.</li>
	<li>Plate four wells of each Salmonella strain. Set up three separate plates for one set of phagocytic cells invasion assay, and mark them with &#8220;association&#8221;, &#8220;invasion&#8221; and &#8220;replication&#8221;, respectively.</li>
	<li>Place the plates in the 5% CO2 incubator at 37 &#176;C ON to allow the macrophage cells to attach to the bottom of the wells.</li>
</ol>
2. Preparation of Salmonella Wild-type and Mutants
<ol>
	<li>On the same day of preparing the macrophage RAW264.7 cells, pick single colonies of wild-type (WT) Salmonella enterica serotype Typhimurium 14028s, DinvA mutant, DphoP mutant, and the desired test mutant strains, and culture them in Luria-Bertani (LB) broth supplemented with antibiotics as appropriate. Use each set of the phagocytic cells invasion assays to test up to 10 mutant strains (DinvA mutant and DphoP mutant are defective in invasion and intracellular replication, respectively).</li>
	<li>Grow the bacteria for 14 hr at 37 &#176;C with shaking at 220 rpm in 5 ml each of LB broth in loosely capped 14 ml polypropylene in a round bottom tube. LB broth contains appropriate antibiotics, in our case, most of mutant strains are resistant to Kanamycin, 50 &#181;g/ml</li>
	<li>The next day, subculture each of the ON cultures of Salmonella strains in plain 5 ml of LB broths at a ratio of 1:50 for an additional 4 hr with shaking at 220 rpm.</li>
	<li>Read OD600 values of each bacterial culture on a spectrophotometer, and each read should range from 0.6-1.2 to optimize Salmonella pathogenicity island-1 type III secretion system (SPI-1 TTSS) gene expression for invasion.</li>
	<li>Use the formula of 1 OD600 = 7.5 x 108 CFU/ml to estimate the bacterial concentration, then dilute bacteria to a concentration of 5 x 106 CFU/ml in fresh DMEM cell culture medium.</li>
</ol>
3. Invasion Assay in 96-well Culture Plate
<ol>
	<li>Remove three 96-well cell culture plates from the CO2 incubator and wash each well once with 200 &#956;l of 1x PBS buffer.</li>
	<li>After washing and complete removal of PBS, add 200 &#956;l bacteria in DMEM medium (see above) into each well. This results in 1 x&#160;106 Salmonella cells in each well and multiplicity of infection (MOI) of 20:1.</li>
	<li>Centrifuge the plates at 1,000 x&#160;g in a sealed carrier for 10 min, then replace plates (time zero) in a 37 &#176;C, 5% CO2 incubator for 30 min. For each strain, set up at least four duplicate wells.</li>
	<li>After 30 min, remove plates from the incubator and wash three times with 200 &#956;l&#160;of 1x PBS to remove unassociated bacteria.</li>
	<li>Following washing, save the plate labeled with &#8220;association&#8221; for further treatment. Add 200 &#956;l of DMEM medium containing 100 &#956;g/ml Gentamicin to each well of the plates marked with &#8220;invasion&#8221; and &#8220;replication&#8221; in order to kill the extracellular Salmonella. Return the plates to 37 &#176;C, 5% CO2 incubator for an additional hr.</li>
	<li>Save one well from each infected strain for recording macrophage cell count, treat the rest of wells on the &#8220;association&#8221; plate with 200 &#956;l of 1x PBS containing 1% Triton X-100 for 10 min. The Triton solution will lyse macrophage cells and release associated Salmonella.</li>
	<li>Harvest Salmonella from each well and place them into 1.5 ml Eppendorf tubes. Perform three 10 fold serial dilutions with 900&#181;l 1x PBS on the harvested samples from each well, vortex is performed between each dilution.</li>
	<li>Plate the third dilution, 10-3, on either LB (WT) or LB Kan (mutants) plates. Label the plates with appropriate Salmonella strain name, dilution number, time point and date.</li>
	<li>After vortexing the third dilution tube, dispense 10 &#181;l sample on the surface of the agar and repeat four times for a total of 5 drops. Be sure to space the five 10 &#956;l drops evenly on the Petri dish plates7.</li>
	<li>After the drops soak into the agar, turn the plates over and incubate overnight at 37 &#176;C, 5% CO2&#160;incubator.</li>
	<li>Treat wells saved for counting of macrophages with 150 &#181;l 1xPBS containing 0.25% Trypsin-EDTA for 10 min.</li>
	<li>After trypsinization, place macrophages into 1.5 ml Eppendorf tubes and treat them with 50 &#181;l of FBS to neutralize Trypsin/EDTA solution, stain the cells with 0.4% Trypan blue and score them through using hemacytometer.</li>
	<li>When the one hr incubation has elapsed, remove the &#8220;invasion&#8221; and &#8220;replication&#8221; plates from the CO2 incubator, and wash each well as &#8220;Step 3.4&#8221;.</li>
	<li>Save the &#8220;invasion&#8221; plate for further treatment as &#8220;Step 3.6-3.12&#8221;.</li>
	<li>Add 200 &#181;l of DMEM medium containing 10 &#956;g/ml Gentamicin to the &#8220;replication&#8221; plate to maintain clearance of extracellular Salmonella in the medium. Return the plate to 37 &#176;C, 5% CO2 incubator for an additional 22.5 hr.</li>
	<li>The next day, remove the &#8220;replication&#8221; plate from 37 &#176;C, 5% CO2 incubator, and wash each well as &#8220;Step 3.4&#8221; and treat them as &#8220;Step 3.6-3.12&#8221;.</li>
	<li>Lastly, remove the agar plates after incubation overnight in 37 &#176;C incubator and score the number of bacterial colonies.</li>
	<li>A good colony distribution should be between 10 and 100 colonies, each spot approximately contains 2 to 20 colonies,&#160;it would be hard to score if there were more than 20 colonies on one spot. If the count is too low or too high, replate the sample with 10-fold higher or lower dilutions as needed.</li>
</ol>
4. Data Analysis
<ol>
	<li>Calculate the CFU (colony formation units per ml) of each plate based on the plating volume and dilution factor.</li>
	<li>Ascertain the number of Salmonella per macrophage through the recorded macrophage cell count from each strain.</li>
	<li>Calculate the geometric means of CFU per macrophage from at least three independent experiments for cell association, invasion or replication, respectively, and normalize further to the WT for the relative value.</li>
	<li>Analyze the data for association, invasion, and replication by Student&#8217;s t-test respectively, by comparing each strain to WT, and determine the statistical significance by the p-value.</li>
</ol>

<ol>
	<li>Haraga, A., Ohlson, M. B., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonellae+interplay+with+host+cells.">Salmonellae interplay with host cells.</a> Nat Rev Microbiol. 6, 53-66 (2008).</li><li>Mandell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+intraleukocytic+bacteria+and+antibiotics.">Interaction of intraleukocytic bacteria and antibiotics.</a> The Journal of clinical investigation. 52, 1673-1679 (1973).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-420 (1994).</li><li>Elsinghorst, E. A., Weitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+invasion+and+adherence+directed+by+the+enterotoxigenic+Escherichia+coli+tib+locus+is+associated+with+a+104-kilodalton+outer+membrane+protein.">Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein.</a> Infect Immun. 62, 3463-3471 (1994).</li><li>Behlau, I., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PhoP-repressed+gene+promotes+Salmonella+typhimurium+invasion+of+epithelial+cells.">A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells.</a> J Bacteriol. 175, 4475-4484 (1993).</li><li>Hueck, C. 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=Salmonella+typhimurium+secreted+invasion+determinants+are+homologous+to+Shigella+Ipa+proteins.">Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins.</a> Mol Microbiol. 18, 479-490 (1995).</li><li>Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+epithelial+cells+with+Salmonella+enterica.">Infection of epithelial cells with Salmonella enterica.</a> Methods Mol Biol. 431, 201-211 (2008).</li><li>Foster, J. W., Spector, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Salmonella+survive+against+the+odds.">How Salmonella survive against the odds.</a> Annu Rev Microbiol. 49, 145-174 (1995).</li><li>Edwards, A. M., Massey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+cells+by+a+bacterial+pathogen.">Invasion of human cells by a bacterial pathogen.</a> Journal of visualized experiments JoVE. 10, (2011).</li><li>Molinari, G., 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=The+role+played+by+the+group+A+streptococcal+negative+regulator+Nra+on+bacterial+interactions+with+epithelial+cells.">The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.</a> Mol Microbiol. 40, 99-114 (2001).</li><li>Van der Velden, A. W., Lindgren, S. W., Worley, M. J., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+pathogenicity+island+1-independent+induction+of+apoptosis+in+infected+macrophages+by+Salmonella+enterica+serotype+typhimurium.">Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype typhimurium.</a> Infect Immun. 68, 5702-5709 (2000).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet Pathol. 39, 200-215 (2002).</li><li>Lawhon, S. 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=Role+of+SPI-1+secreted+effectors+in+acute+bovine+response+to+Salmonella+enterica+Serovar+Typhimurium+a+systems+biology+analysis+approach.">Role of SPI-1 secreted effectors in acute bovine response to Salmonella enterica Serovar Typhimurium a systems biology analysis approach.</a> PLoS One. 6, e26869 (2011).</li><li>Drecktrah, D., Knodler, L. A., Galbraith, K., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Salmonella+SPI1+effector+SopB+stimulates+nitric+oxide+production+long+after+invasion.">The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion.</a> Cell Microbiol. 7, 105-113 (2005).</li></ol>

We have nothing to disclose.

The Gentamicin protection assay is widely used to study the invasion and replication of intracellular bacterial pathogens inside host cell, and it is especially an important biological tool for studying pathogens, like Salmonella, whose invasion is the prerequisite step for establishing infection1. The standard Gentamicin protection assay in Salmonella research community is implemented in 24-well culture dish5. Though the use of 48 or even 96-well plates were discussed before for h...

Nontyphoidal Salmonella are important causes of enteric diseases in all vertebrates. Salmonellosis in humans is among the top bacterial food-borne diseases1. Characterization of the molecular mechanisms that underpin the interactions of Salmonella with their animal hosts is mainly achieved through the study of Salmonella enterica serotype Typhimurium (STM) in tissue culture and animal models of infection. Gaining insights in STM-host interactions will help us understand how Salmonella survive and grow inside host cells. The first challenge in studying these interactions is to identify as many participating factors as possible from both host and pathogen, but these endeavors are largely obstructed by the significant difficulties of dealing with two independent complex biological systems simultaneously, i.e., host and Salmonella, under physiological conditions. Additionally, the large repertoire of Salmonella and host genes potentially encoding factors involved in host interactions require high-throughput biological platform to tackle this challenge.
A modified, standardized assay to phenotype Salmonella association, invasion&#160;and replication in macrophages with high-throughput capacity was developed to examine a large set of genes likely engaging in Salmonella-host interactions. The Gentamicin protection assay was developed in 19732, but was first thoroughly described by Elsinghorst in 19943,4. It has now become a standard tool for studying many intracellular bacterial pathogens ex vivo, including Salmonella5,6. Internalized bacteria avoid being killed by some antibiotics, like Gentamicin, that cannot penetrate eukaryotic cells3. By taking advantage of this phenomenon, the Gentamicin protection assay measures the survival and growth of intracellular bacterial pathogens. Three events during the infection, i.e., association with eukaryotic cells, invasion and replication, can be evaluated for intracellular bacterial pathogens based on the time interval between infection, Gentamicin treatment, and further incubation (Figure&#160;1). Eukaryotic cell lines provide a physiological environment that is less complex than relevant animal models for host-pathogen interaction studies.
The Gentamicin protection assay is an appropriate platform to study STM-host interactions, but the standard assay in a 24-well culture dish has low-throughput capacity. Computational analysis of in vivo datasets identified 149 Salmonella gene products that are predicted to interact with approximately 300 host gene products (unpublished data). The standard Gentamicin protection assay does not have the capacity to phenotype this number of mutants efficiently.
In addition, the Gentamicin protection assay can theoretically detect the invasion of even a single bacterium. Because of this inherent sensitivity, the raw data are susceptible to technical variances when repeated at different times. The internal controls and relative data presentation after normalization are essential for meaningful interpretation of the results. Given these considerations, a modified, standardized Gentamicin protection assay was developed to enhance testing capacity and increase precision.
The following protocol is detailed and illustrated to perform the modified Gentamicin protection assay using 96-well culture dishes and the murine macrophage RAW264.7 cell line. Compared to the standard protocol in 24-well culture dishes, the modified protocol has the following advantages: 1) Using 96-well culture dishes allows up to 10 different mutant strains to be phenotyped including internal positive and negative controls with sufficient statistical power; 2) The variance of results is significantly reduced, because the mutant strains are tested in the same culture dish and at the same time; 3) The use of multichannel pipettes increases throughput while reducing operator fatigue. Lastly, comparing to 24-well culture dishes, concerns of less host cells per well in 96-well culture dish were addressed through protocol optimization and standardization.
In summary, the modified, standardized assay to in vitro phenotype Salmonella or other bacterial association, invasion and replication in phagocytic cells increases precision and achieves high-throughput capacity while reducing operator fatigue.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:173px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">11965</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Fetal bovine serum</td>
			<td style="width:173px;">HyClone</td>
			<td style="width:173px;">SH30910.03</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">T-75 cell culture flask vented filter cap</td>
			<td style="width:173px;">Nest Biotechnology</td>
			<td style="width:173px;">708003</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">100X Non-Essential Amino Acids</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">11140</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Cell scraper</td>
			<td style="width:173px;">BD Falcon</td>
			<td style="width:173px;">353086</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">96-well cell culture plate</td>
			<td style="width:173px;">Corning Incorporated</td>
			<td style="width:173px;">3595</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">Luria-Bertani (LB) broth</td>
			<td style="width:173px;">MP Biomedicals</td>
			<td style="width:173px;">3002-075</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:173px;">BD Falcon</td>
			<td style="width:173px;">352059</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">PBS pH7.4 (1x)</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">10010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Triton X-100</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Kanamycin solution</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">K0254</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Gentamicin solution</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">G1272</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">0.25% Trypsin-EDTA</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">25200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Trypan blue</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

See representative results (Figure 2) after the data are plotted based on the modified phagocytic cell invasion assay. The data include five different strains, WT, &#916;invA, &#916;phoP, mutant A, and mutant B. &#916;invA,&#160;known to be defective for invasion, and &#916;phoP known to be defective for replication8, are used as positive controls to assess the experimental validity. Indeed, in the modified invasion assay, a &#916;invA mutant is int...

Watch this Scientific Journal Video about High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages at JoVE.com

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.

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 ...

high-throughput-assay-to-phenotype-salmonella-enterica-typhimurium

Research

JoVE Journal

Immunology and Infection

12.5K Views.  Texas A&M University. 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.

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

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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 ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...