Quantification of Candida albicans by flow cytometry using TO-PRO®-3 iodide as a single-stain viability dye

Kerstens, Monique; Boulet, Gaëlle; Pintelon, Isabel; Hellings, Mario; Voeten, Liesbeth; Delputte, Peter; Maes, Louis; Cos, Paul

doi:10.1016/j.mimet.2012.12.006

Cited by 10 publications

(11 citation statements)

References 10 publications

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“…Promastigotes at different phases in their growth curve were counted by FCM and used to infect primary peritoneal mouse macrophages, adopting a 15/1 parasite/macrophage ratio. To correct for the variable number of dead promastigotes, live/dead staining was carried out using the single-stain viability dye TO-PRO®-3 iodide (Molecular probes®, OR, USA) [ 30 ]. Promastigote uptake was enhanced by reducing the culture volume to 30 μl and macrophages were incubated at 37°C in 5% CO 2 for 4 hours, after which 100 μl of macrophage medium was added to each well.…”

Section: Methodsmentioning

confidence: 99%

Comparative Fitness of a Parent Leishmania donovani Clinical Isolate and Its Experimentally Derived Paromomycin-Resistant Strain

et al. 2015

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Paromomycin has recently been introduced for the treatment of visceral leishmaniasis and emergence of drug resistance can only be appropriately judged upon its long term routine use in the field. Understanding alterations in parasite behavior linked to paromomycin-resistance may be essential to assess the propensity for emergence and spread of resistant strains. A standardized and integrated laboratory approach was adopted to define and assess parasite fitness of both promastigotes and amastigotes using an experimentally induced paromomycin-resistant Leishmania donovani strain and its paromomycin-susceptible parent wild-type clinical isolate. Primary focus was placed on parasite growth and virulence, two major components of parasite fitness. The combination of in vitro and in vivo approaches enabled detailed comparison of wild-type and resistant strains for which no differences could be demonstrated with regard to promastigote growth, metacyclogenesis, in vitro infectivity, multiplication in primary peritoneal mouse macrophages and infectivity for Balb/c mice upon infection with 2 x 107 metacyclic promastigotes. Monitoring of in vitro intracellular amastigote multiplication revealed a consistent decrease in parasite burden over time for both wild-type and resistant parasites, an observation that was subsequently also confirmed in a larger set of L. donovani clinical isolates. Though the impact of these findings should be further explored, the study results suggest that the epidemiological implications of acquired paromomycin-resistance may remain minimal other than the loss of one of the last remaining drugs effective against visceral leishmaniasis.

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Section: Methodsmentioning

confidence: 99%

Comparative Fitness of a Parent Leishmania donovani Clinical Isolate and Its Experimentally Derived Paromomycin-Resistant Strain

et al. 2015

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“…Quantification of intracellular MIL accumulation was achieved by measuring fluorescence intensity in the FL2-channel by flow cytometry (FACSCalibur ® ). TO-PRO ® -3 iodide (Molecular Probes ® , Eugene, OR, USA) was used for live/dead staining and 100,000 events per sample were recorded [21]. Nontreated parasites were included as negative control.…”

Section: Evaluation Of Mil Uptake Ratesmentioning

confidence: 99%

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection

et al. 2020

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Background: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. Methods: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. Results: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. Conclusions: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.

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“…8 The viable and dead cultures were analyzed separately to confirm the localization of the respective populations in the FCM dotplot and to set the gates that delineate these groups.…”

Section: Qualitative Analysismentioning

confidence: 99%

“…[4][5][6][7] In a previous article, we reported the use of an FCM single-stain approach to directly quantify viable Candida albicans cells. 8 Viable cells were discriminated from dead cells through a difference in fluorescence intensity using TO-PRO ® -3 iodide (TP3), a membrane-impermeable dye that shows a greatly enhanced fluorescence on binding to double-stranded DNA (dsDNA) in bacteria that are dead and thus permeable.…”

Section: Introductionmentioning

confidence: 99%

Flow Cytometric Enumeration of Bacteria Using TO-PRO®-3 Iodide as a Single-Stain Viability Dye

et al. 2014

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Quantification of bacteria using conventional viable plate counting (VPC) is labor-intensive and time-consuming. Flow cytometry (FCM) can be proposed as a faster alternative. This study aimed to develop a flow cytometric, single-stain approach using TO-PRO ® -3 iodide (TP3) for the quantification of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis cells. Live or dead bacterial suspensions were stained with TP3 and analyzed using a FACSCalibur flow cytometer. After optimization of staining parameters and instrument settings, an excellent separation of viable and dead cells was achieved for all species. The quantitative performance of the technique was assessed by analyzing serial dilutions of bacterial suspensions using FCM and VPC. A highly linear correlation (r 2 > 0.99) was observed between the colony forming units (CFU)/mL as determined by FCM and by VPC over a concentration range of about 10 4 to 10 8 CFU/mL. As such, FCM quantification of viable bacteria using TP3 can be considered as an accurate and reliable alternative for VPC. The monostain procedure is easy to apply and cost-effective, and it allows bacterial enumeration in a broad variety of samples.

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Quantification of Candida albicans by flow cytometry using TO-PRO®-3 iodide as a single-stain viability dye

Cited by 10 publications

References 10 publications

Comparative Fitness of a Parent Leishmania donovani Clinical Isolate and Its Experimentally Derived Paromomycin-Resistant Strain

Comparative Fitness of a Parent Leishmania donovani Clinical Isolate and Its Experimentally Derived Paromomycin-Resistant Strain

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection

Flow Cytometric Enumeration of Bacteria Using TO-PRO®-3 Iodide as a Single-Stain Viability Dye

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