Simple Resazurin-Based Microplate Assay for Measuring Chlamydia Infections

Osaka, Ichie; Hefty, P. Scott

doi:10.1128/aac.00056-13

Cited by 33 publications

(33 citation statements)

References 19 publications

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“…Prior to introducing pASK-GFP-L2 into C. trachomatis , sensitivity to the antibiotic ATc was evaluated given the relatively high sensitivity of Chlamydia to tetracycline [24,25]. In other diverse bacteria ( Mycobacterium , Borrelia , E. coli , and Staphyloccus ), final concentrations of ATc ranging from 0.25 to 20.0 µg/ml induced gene expression without apparent negative effects [9-12].…”

Section: Resultsmentioning

confidence: 99%

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

et al. 2013

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Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.

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Section: Resultsmentioning

confidence: 99%

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

et al. 2013

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“…Our method was capable of counting 1:2 dilutions of inclusions of three different chlamydial species and provided a high correlation with the theoretical estimates. The system was capable of measuring the inclusion counts over a 1-log-unit range, which is comparable to or better than that of other previously described methods (12,(23)(24)(25). As with the other methods, a limitation of the ChlamyCount method at higher MOIs is the accurate dissection of the high number of confluent or nearly confluent fluorescent areas that originate from close inclusions.…”

Section: Discussionmentioning

confidence: 74%

“…The first approach uses either a fluorimeter or a spectrophotometer to measure the total intensity of a Chlamydia-specific fluorescently labeled antibody (26) or indirectly measure Chlamydia growth by measuring decreased host cell metabolism after Chlamydia-induced lysis (25). These methods are rapid and inexpensive but do not rely on counting the individual inclusions; hence, the possibility of aspecific antibody binding or an aspecific change in host cell metabolism cannot be excluded.…”

Section: Discussionmentioning

confidence: 99%

Application of DNA Chip Scanning Technology for Automatic Detection of Chlamydia trachomatis and Chlamydia pneumoniae Inclusions

Bogdanov

Endrész

Urbán

et al. 2014

Antimicrob Agents Chemother

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e Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High-or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions.

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“…This assay can also be modified for use in a high throughput screen with the inclusion of viability indicators such as alamarBlue (Osaka and Hefty, 2013). Notably, this assay will likely increase our knowledge related to the regulation of ICP0’s transactivating activity, which may eventually lead to potential treatments against HSV-1 and its recurrent diseases.…”

Section: Discussionmentioning

confidence: 99%

Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0

2015

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The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-hour period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0.

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Simple Resazurin-Based Microplate Assay for Measuring Chlamydia Infections

Cited by 33 publications

References 19 publications

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

Application of DNA Chip Scanning Technology for Automatic Detection of Chlamydia trachomatis and Chlamydia pneumoniae Inclusions

Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0

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