A Novel Flow Cytometric Approach for the Quantification and Quality Control of Chlamydia trachomatis Preparations

Chlamydia trachomatis is an obligate intracellular pathogenic bacterium with a biphasic developmental cycle manifesting two distinct morphological forms: infectious elementary bodies (EBs) and replicative intracellular reticulate bodies (RBs). Current standard protocols for quantification of the isolates assess infectious particles by titering inclusion-forming units, using permissive cell lines, and analyzing via immunofluorescence. Enumeration of total particle counts is achieved by counting labeled EBs/RBs using a fluorescence microscope. Both methods are time-consuming with a high risk of observer bias. For a better assessment of C. trachomatis preparations, we developed a simple and time-saving flow cytometry-based workflow for quantifying small particles, such as EBs with a size of 300 nm. This included optimization of gain and threshold settings with the addition of a neutral density filter for small-particle discrimination. The nucleic acid dye SYBR® Green I (SGI) was used together with propidium iodide and 5(6)-carboxyfluorescein diacetate to enumerate and discriminate between live and dead bacteria. We found no significant differences between the direct particle count of SGI-stained C. trachomatis preparations measured by microscopy or flow cytometry (p > 0.05). Furthermore, we completed our results by introducing a cell culture-independent viability assay. Our measurements showed very good reproducibility and comparability to the existing state-of-the-art methods, indicating that the evaluation of C. trachomatis preparations by flow cytometry is a fast and reliable method. Thus, our method facilitates an improved assessment of the quality of C. trachomatis preparations for downstream applications.

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Quantitative Monitoring of the Chlamydia trachomatis Developmental Cycle Using GFP-Expressing Bacteria, Microscopy and Flow Cytometry

PLoS ONE ◽

10.1371/journal.pone.0099197 ◽

2014 ◽

Vol 9 (6) ◽

pp. e99197 ◽

Cited By ~ 20

Author(s):

François Vromman ◽

Marc Laverrière ◽

Stéphanie Perrinet ◽

Alexandre Dufour ◽

Agathe Subtil

Keyword(s):

Flow Cytometry ◽

Chlamydia Trachomatis ◽

Developmental Cycle ◽

Quantitative Monitoring

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Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

Scientific Reports ◽

10.1038/s41598-020-79831-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Weichao Zhai ◽

Jerome Tan ◽

Tobias Russell ◽

Sixun Chen ◽

Dennis McGonagle ◽

...

Keyword(s):

Flow Cytometry ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Preclinical Model ◽

Label Free ◽

Current Standard ◽

Differentiation Potential ◽

Human Mesenchymal Stromal Cells ◽

Endogenous Fluorophores

AbstractHuman mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

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Effects ofMentha suaveolensEssential Oil onChlamydia trachomatis

BioMed Research International ◽

10.1155/2015/508071 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Rosa Sessa ◽

Marisa Di Pietro ◽

Fiorenzo De Santis ◽

Simone Filardo ◽

Rino Ragno ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Substantial Reduction ◽

Developmental Cycle ◽

Elementary Body ◽

Promising Candidate ◽

Common Cause ◽

Sexually Transmitted ◽

Reticulate Body ◽

Preventative Strategy

Chlamydia trachomatis, the most common cause of sexually transmitted bacterial infection worldwide, has a unique biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body.C. trachomatisis responsible for severe reproductive complications including pelvic inflammatory disease, ectopic pregnancy, and obstructive infertility. The aim of our study was to evaluate whetherMentha suaveolensessential oil (EOMS) can be considered as a promising candidate for preventingC. trachomatisinfection. Specifically, we investigated thein vitroeffects of EOMS towardsC. trachomatisanalysing the different phases of chlamydial developmental cycle. Our results demonstrated that EOMS was effective towardsC. trachomatis, whereby it not only inactivated infectious elementary bodies but also inhibited chlamydial replication. Our study also revealed the effectiveness of EOMS, in combination with erythromycin, towardsC. trachomatiswith a substantial reduction in the minimum effect dose of antibiotic. In conclusion, EOMS treatment may represent a preventative strategy since it may reduceC. trachomatistransmission in the population and, thereby, reduce the number of new chlamydial infections and risk of developing of severe sequelae.

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A novel consistent photomicrography technique using a reference slide made of neutral density filter

Microscopy Research and Technique ◽

10.1002/jemt.20921 ◽

2010 ◽

Vol 74 (5) ◽

pp. 397-400 ◽

Cited By ~ 1

Author(s):

Jong Yeob Kim ◽

Ji Woong Kim ◽

Soo Hong Seo ◽

Young Chul Kye ◽

Hyo Hyun Ahn

Keyword(s):

Neutral Density ◽

Neutral Density Filter ◽

Density Filter

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Chlamydial infection and disease in the koala

Microbiology Australia ◽

10.1071/ma05065 ◽

2005 ◽

Vol 26 (2) ◽

pp. 65 ◽

Cited By ~ 3

Author(s):

Peter Timms

Keyword(s):

Chlamydia Trachomatis ◽

Chlamydial Infection ◽

Developmental Cycle ◽

Elementary Body ◽

Obligate Intracellular ◽

Molecular Approaches ◽

Reticulate Body ◽

Wide Range ◽

The Family ◽

Serious Disease

Chlamydiae are obligate intracellular bacterial pathogens able to infect and cause serious disease in humans, birds and a remarkably wide range of warm and cold-blooded animals. The family Chlamydiaciae have traditionally been defined by their unique biphasic developmental cycle, involving the interconversion between an extracellular survival form, the elementary body and an intracellular replicative form, the reticulate body. However, as with many other bacteria, molecular approaches including 16SrRNA sequence are becoming the standard of choice. As a consequence, the chlamydiae are in a taxonomic state of flux. Prior to 1999, the family Chlamydiaceae consisted of one genus, Chlamydia, and four species, Chlamydia trachomatis, C. psittaci, C. pecorum and C. pneumoniae. In 1999, Everett et al proposed a reclassification of Chlamydia into two genera (Chlamydia and Chlamydophila) and nine species (Chlamydia trachomatis, C. suis, and C. muridarum and Chlamydophila psittaci, C. pneumoniae, C. felis, C. pecorum, C. abortus, and C. caviae). While some of these species are thought to be host specific (C. suis ? pigs, C. muridarum ? mice, C. felis ? cats, C. caviae ? guinea pigs) many are known to infect and cause disease in a wide range of hosts.

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Transcriptional Expression of the ompA, cpaf, tarp, and tox Genes of Chlamydia trachomatis Clinical Isolates at Different Stages of the Developmental Cycle

Microorganisms ◽

10.3390/microorganisms7060153 ◽

2019 ◽

Vol 7 (6) ◽

pp. 153 ◽

Cited By ~ 4

Author(s):

Suvi Korhonen ◽

Kati Hokynar ◽

Laura Mannonen ◽

Jorma Paavonen ◽

Eija Hiltunen-Back ◽

...

Keyword(s):

Gene Expression ◽

Chlamydia Trachomatis ◽

Cultured Cells ◽

Expression Patterns ◽

Clinical Isolates ◽

Gene Expression Patterns ◽

Developmental Cycle ◽

Passage Number ◽

Low Passage ◽

Reference Strains

The transcriptional gene expression patterns of Chlamydia trachomatis have mainly been studied using reference strains propagated in cultured cells. Here, using five low-passage-number C. trachomatis clinical isolates that originated from asymptomatic or symptomatic female patients, the in vitro expression of the ompA, cpaf, tarp, and tox genes was studied with reverse transcriptase real-time PCR during the chlamydial developmental cycle. We observed dissimilarities in the gene expression patterns between the low-passage-number clinical isolates and the reference strains. The expression of ompA and the peak of the tox expression were observed earlier in the reference strains than in most of the clinical isolates. The expression of cpaf was high in the reference strains compared with the clinical isolates at the mid-phase (6–24 hours post infection) of the developmental cycle. All of the strains had a rather similar tarp expression profile. Four out of five clinical isolates exhibited slower growth kinetics compared with the reference strains. The use of low-passage-number C. trachomatis clinical isolates instead of reference strains in the studies might better reflect the situation in human infection.

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Expression, Processing, and Localization of PmpD of Chlamydia trachomatis Serovar L2 during the Chlamydial Developmental Cycle

PLoS ONE ◽

10.1371/journal.pone.0000568 ◽

2007 ◽

Vol 2 (6) ◽

pp. e568 ◽

Cited By ~ 26

Author(s):

Andrey O. Kiselev ◽

Walter E. Stamm ◽

John R. Yates ◽

Mary F. Lampe

Keyword(s):

Chlamydia Trachomatis ◽

Developmental Cycle ◽

Expression Processing

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Staining of Chlamydia trachomatis elementary bodies: A suitable method for identifying infected human monocytes by flow cytometry

Journal of Microbiological Methods ◽

10.1016/j.mimet.2006.12.013 ◽

2007 ◽

Vol 69 (1) ◽

pp. 116-121 ◽

Cited By ~ 10

Author(s):

Karen Schnitger ◽

Florence Njau ◽

Ulrike Wittkop ◽

Andrea Liese ◽

Jens G. Kuipers ◽

...

Keyword(s):

Flow Cytometry ◽

Chlamydia Trachomatis ◽

Suitable Method ◽

Human Monocytes

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Influence of Peripheral on Central Stereopsis: An Induced Depth Tilt Effect

Perception ◽

10.1068/v970047 ◽

1997 ◽

Vol 26 (1_suppl) ◽

pp. 276-276

Author(s):

S Müller ◽

E R Wist

Keyword(s):

Opposite Direction ◽

Rotation Speed ◽

Neutral Density ◽

Neutral Density Filter ◽

Disk Rotation ◽

Optimal Rotation ◽

Density Filter ◽

Pulfrich Effect ◽

Variable Errors ◽

Tilted Disk

A large rotating black/white sectored disk (58 deg diameter) viewed with a neutral density filter over one eye is perceived as tilted in depth according to the Pulfrich phenomenon. But with fixation on a centrally located vertical bar (7 deg in length), the disk is perceived as vertical while the central bar is perceived as tilted in the opposite direction. This effect remains even if the central 38 deg portion of the disk is occluded leaving a peripheral annulus 10 deg in width. At an optimal rotation speed of 45° s−1 and a filter of 2 log units, the inter-individual perceived tilt of the bar ranges between 5° and 10° as measured by nulling out the illusory tilt by adjustment with a joystick. Variable errors were extremely small and corresponded well with central stereoscopic resolution. The amount of illusory tilt depends on the speed of disk rotation and filter density, and its direction on the relation between the direction of motion and the filter-covered eye. The effect is not limited to Pulfrich-induced stereotilt: When the disk was stationary but physically tilted in depth, the induced tilt on the central bar corresponded to about 50% of the physical tilt. This effect, in turn, could be cancelled or enhanced by rotating the tilted disk and inducing an appropriate Pulfrich effect. With monocular viewing no induced depth tilt occurs. The results are interpreted in terms of a stereoscopic induced effect operating beyond the known peripheral limits of stereopsis.

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Characterization of Outer Membrane Proteins in Chlamydia trachomatis LGV Serovar L2

Journal of Bacteriology ◽

10.1128/jb.183.8.2686-2690.2001 ◽

2001 ◽

Vol 183 (8) ◽

pp. 2686-2690 ◽

Cited By ~ 38

Author(s):

Regina J. Tanzer ◽

Thomas P. Hatch

Keyword(s):

Chlamydia Trachomatis ◽

Membrane Proteins ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Outer Membrane Proteins ◽

Major Outer Membrane Protein ◽

Developmental Cycle ◽

Reading Frame

ABSTRACT We used a photoactivatable, lipophilic reagent, 3′-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies ofChlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.

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