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  • 1.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bacterial Competition Assay Based on Extracellular D-amino Acid Production2018Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 8, nr 7, artikel-id e2787Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments. With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.

  • 2.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cordier, Baptiste
    van Teeffelen, Sven
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography2020Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 10, nr 19, artikel-id e3780Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry (MS) of the UPLC resolved muropeptides, thus facilitating their identification. This method improves our capability to perform high throughput analysis to better understand the cell- wall biology.

  • 3.
    Bag, Pushan
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Schröder, Wolfgang P.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jansson, Stefan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Farci, Domenica
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Solubilization method for isolation of photosynthetic mega- And super-complexes from conifer thylakoids2021Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 11, nr 17, artikel-id e4144Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of biophysical and biochemical techniques allowed detailed studies of the thylakoid organization and the interaction between protein complexes and cofactors. These studies have mainly focused on model plants, such as Arabidopsis, pea, spinach, and tobacco, which are grown in climate chambers even though significant differences between indoor and outdoor growth conditions are present. In this manuscript, we present a new mild-solubilization procedure for use with “fragile” samples such as thylakoids from conifers growing outdoors. Here, the solubilization protocol is optimized with two detergents in two species, namely Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). We have optimized the isolation and characterization of PSI and PSII multimeric mega- and super-complexes in a close-to-native condition by Blue-Native gel electrophoresis. Eventually, our protocol will not only help in the characterization of photosynthetic complexes from conifers but also in understanding winter adaptation.

  • 4.
    Cervantes-Rivera, Ramón
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Whole-Genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria2020Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 10, nr 18, artikel-id e3757Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5′-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis.Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

  • 5.
    Cheregi, Otilia
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Funk, Christiane
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Antibiotic Disc Assay for Measuring Cell Wall Function in Synechocystis sp. PCC68032016Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 6, nr 24Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This protocol describes how to investigate the integrity of the outer cell wall in thecyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test(Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibioticswere placed on agar plates inoculated with bacteria. The antibiotics we tested, interfering with thebiosynthesis/function of bacterial cell walls, will diffuse into the agar and produce a zone ofcyanobacterial growth inhibition around the disc(s). The size of the inhibition zone reflects the sensitivityof the strain to the action of antibiotics, e.g., a mutation in a protein functioning within the cell wall or itsconstruction would render the mutant strain more sensitive to the respective antibiotic. The method hasproven to be useful for phenotyping a mutant of Synechocystis sp. PCC6803 lacking all three genesencoding Deg proteases. Deletion of these ATP-independent serine proteases was shown to haveimpact on the outer cell layers of Synechocystis cells.

  • 6.
    Hao, Jingfang
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Malnoë, Alizée
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    A simple sonication method to isolate the chloroplast lumen in Arabidopsis thaliana2023Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 13, nr 15, artikel-id e4756Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The chloroplast lumen contains at least 80 proteins whose function and regulation are not yet fully understood. Isolating the chloroplast lumen enables the characterization of the lumenal proteins. The lumen can be isolated in several ways through thylakoid disruption using a Yeda press or sonication, or through thylakoid solubilization using a detergent. Here, we present a simple procedure to isolate thylakoid lumen by sonication using leaves of the plant Arabidopsis thaliana. The step-by-step procedure is as follows: thylakoids are isolated from chloroplasts, loosely associated thylakoid surface proteins from the stroma are removed, and the lumen fraction is collected in the supernatant following sonication and centrifugation. Compared to other procedures, this method is easy to implement and saves time, plant material, and cost. Lumenal proteins are obtained in high quantity and purity; however, some stromal membrane-associated proteins are released to the lumen fraction, so this method could be further adapted if needed by decreasing sonication power and/or time.

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  • 7.
    Håglin, Sofia
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bohm, Staffan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Berghard, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Single or Repeated Ablation of Mouse Olfactory Epithelium by Methimazole2021Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 11, nr 8, artikel-id e3983Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Odor-detecting olfactory sensory neurons residing in the nasal olfactory epithelium (OE) are the only neurons in direct contact with the external environment. As a result, these neurons are subjected to chemical, physical, and infectious insults, which may be the underlying reason why neurogenesis occurs in the OE of adult mammals. This feature makes the OE a useful model for studying neurogenesis and neuronal differentiation, with the possibility for systemic as well as local administration of various compounds and infectious agents that may interfere with these cellular processes. Several different chemical compounds have been shown to cause toxic injury to the OE, which can be used for OE ablation. We, and others, have found that the systemic administration of the hyperthyroid drug, methimazole, reliably causes olfactotoxicity as a side effect. Here, we outline an OE lesioning protocol for single or repeated ablation by methimazole. A single methimazole administration can be used to study neuroepithelial regeneration and stem cell activation, while repeated ablation and regeneration of OE enable the study of tissue stem cell exhaustion and generation of tissue metaplasia.

  • 8.
    Libby, Eric
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik. IceLab.
    Lind, Peter A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Probabilistic models for predicting mutational routes to new adaptive phenotypes2019Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 9, nr 20, artikel-id 3407Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Understanding the translation of genetic variation to phenotypic variation is a fundamental problem in genetics and evolutionary biology. The introduction of new genetic variation through mutation can lead to new adaptive phenotypes, but the complexity of the genotype-to-phenotype map makes it challenging to predict the phenotypic effects of mutation. Metabolic models, in conjunction with flux balance analysis, have been used to predict evolutionary optimality. These methods however rely on large scale models of metabolism, describe a limited set of phenotypes, and assume that selection for growth rate is the prime evolutionary driver.

    Here we describe a method for computing the relative likelihood that mutational change will translate into a phenotypic change between two molecular pathways. The interactions of molecular components in the pathways are modeled with ordinary differential equations. Unknown parameters are offset by probability distributions that describe the concentrations of molecular components, the reaction rates for different molecular processes, and the effects of mutations. Finally, the likelihood that mutations in a pathway will yield phenotypic change is estimated with stochastic simulations.

    One advantage of this method is that only basic knowledge of the interaction network underlying a phenotype is required. However, it can also incorporate available information about concentrations and reaction rates as well as mutational biases and mutational robustness of molecular components. The method estimates the relative probabilities that different pathways produce phenotypic change, which can be combined with fitness models to predict evolutionary outcomes.

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  • 9.
    Lopes, Jose Pedro
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Visualizing Hypoxia in a Murine Model of Candida albicans Infection Using in vivo Biofluorencence2019Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 9, nr 15, artikel-id e3326Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Candida albicans is a leading human fungal pathogen that uses several metabolic adaptations to escape immune cells and causes systemic disease. Here, we describe a protocol for measuring one of these adaptations, the ability to thrive in hypoxic niches. Hypoxia was generated after successful subdermal infection with C. albicans in a murine infection model. Hypoxia was measured using a fluorescent dye for carbonic anhydrase 9, a host enzyme active under hypoxic conditions. Emitted fluorescence was subsequently quantified using an IVIS system. This protocol was optimized for the use in subdermal infection in mice but has the potential to be adapted to other models of fungal infection.

  • 10.
    Pastula, Agnieszka
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells2014Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 4, nr 10, artikel-id e1125Artikel i tidskrift (Refereegranskat)
  • 11.
    Pastula, Agnieszka
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Induction of Epithelial-mesenchymal Transition in MDCK II Cells2021Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 11, nr 3, artikel-id e3903Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epithelial-mesenchymal transition (EMT) is a reversible process of epithelial cell transdifferentiation into a mesenchymal cell, that enables initiation of cell migration. EMT plays an important role in embryonic development, tissue repair and cancer metastasis. Better understanding of cellular and molecular events during EMT will not only provide novel insights on how mammalian organism develops and how epithelial tissues regenerate, but also can identify novel therapeutic targets for cancer therapy. Here we aim to provide a detailed protocol on how to induce EMT in Madin-Darby Canine Kidney (MDCK) II epithelial cell line and perform immunofluorescent staining on EMT-induced cells.

  • 12.
    Puhar, Andrea
    et al.
    Inserm U786 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France.
    Sansonetti, Philippe J.
    Microbiologie et Maladies Infectieuses, Collège de France, Paris, France.
    Dye-uptake Experiment through Connexin Hemichannels2014Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 4, nr 17, artikel-id e1221Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    [Abstract] Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al., 2013; Tran Van Nhieu et al., 2003). Hemichannel opening can be measured by electrophysiology, by quantifying the release of a hemichannel-permeable molecule into the extracellular medium or by quantifying the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule. As the extent of uptake of a molecule is proportional to its concentration, exposure time, temperature (these parameters are controlled) and, importantly, to the number of active hemichannels on the cell surface, uptake assays are routinely used to assess hemichannel opening. This protocol for the uptake of the fluorescent dye ethidium bromide was used with Hela cells that were stably transfected with Cx26 or Cx43 (Paemeleire et al., 2000). Nevertheless, it could likely be used with other Cx-expressing cell types.

  • 13.
    Puhar, Andrea
    et al.
    Inserm U786 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France.
    Sansonetti, Philippe J.
    Microbiologie et Maladies Infectieuses, Collège de France, Paris, France.
    Induction of Connexin-hemichannel Opening2014Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 4, nr 17, artikel-id e1220Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    [Abstract] Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al., 2013; Tran Van Nhieu et al., 2003). This protocol was used with epithelial cells, in particular with polarized and non-polarized intestinal epithelial TC7 cells and with Hela cells that were stably transfected with Cx26 or Cx43 (Paemeleire et al., 2000). Nevertheless, it could likely be used with other Cx-expressing cell types. Whether hemichannels are open can be determined by electrophysiology or by measuring the release into the extracellular medium of a hemichannel permeable molecule (for example, ATP) or the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule [for example, the fluorescent dye ethidium bromide-see associated protocol “Dye-uptake Experiment through Connexin Hemichannels” (Puhar and Sansonetti, 2014)].

  • 14.
    Sharma, Atin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri2019Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 9, nr 13, artikel-id e3292Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella flexneri is an intracellular bacterial pathogen that gains access to the gut epithelium using a specialized Type III Secretion System (T3SS). Various determinants mediating this invasive infection have been experimentally verified using the classical gentamicin protection assay presented here. In this assay epithelial cell lines are infected by bacteria in vitro and the extracellular bacteria are killed by gentamicin. The internalized bacteria, which are protected from the bactericidal action of gentamicin, are recovered by lysing the epithelial cells and enumerated by determining the colonies formed on solid medium. Various techniques based on light microscopy, such as immunofluorescence and bacteria expressing fluorescent proteins, are also used for studying intracellular bacteria. However, these techniques are not only labor intensive and require sophisticated equipment, but mostly are also not quantitative. Despite being an easy quantitative method to study invasiveness of bacteria, the gentamicin protection assay cannot distinguish between the survival and multiplication of the internalized bacteria over longer incubation periods. To alleviate the complications created by multiplication and dissemination of internalized bacteria, complementary assays like plaque formation assays are required. This protocol presents an easy and cost-effective method to determine the invasiveness and the capacity to establish an infection of Shigella under different conditions.

  • 15.
    Sharma, Atin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells2019Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 9, nr 13, artikel-id e3293Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella flexneri invades the epithelial cells lining the gut lumen and replicates intracellularly. The specialized Type III Secretion System (T3SS) and its effector proteins, encoded on a large virulence plasmid, assist the bacterium to gain access to the cytosol. Thereafter Shigella disseminates to neighboring cells in an epithelial layer without further extracellular steps. Host cell lysis occurs when these bacteria have extensively replicated in the target cell cytosol. Here we describe a simple method to qualitatively as well as quantitatively study the capacity of Shigella to invade and disseminate within an epithelium by assessing the number and size of plaques representing the dead cells in a monolayer of TC7 cells. This classical protocol follows a simple approach of infecting the monolayers of epithelial cell lines with Shigella and visualizing the dead cells as plaques formed against a stained background.

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