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A new methodology for evaluation of nematode viability.

Ferreira SR, Mendes TA, Bueno LL, de Araújo JV, Bartholomeu DC, Fujiwara RT - Biomed Res Int (2015)

Bottom Line: Furthermore, data on motility test presented an inverse correlation with fluorimetric data when ivermectin was used.Our results showed that lower concentrations of drugs were effective to interfere in the processes of cellular transport while higher drugs concentrations were necessary in order to result in any damage to cell integrity.The methodology described in this work might be useful for studies that aim to evaluate the viability of nematodes, particularly for testing of new anthelminthic compounds using an easy, economic, reproducible, and no time-consuming technique.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil.

ABSTRACT
Nematodes infections are responsible for debilitating conditions and economic losses in domestic animals as well as livestock and are considered an important public health problem due to the high prevalence in humans. The nematode resistance for drugs has been reported for livestock, highlighting the importance for development of new anthelmintic compounds. The aim of the current study was to apply and compare fluorimetric techniques using Sytox and propidium iodide for evaluating the viability of C. elegans larvae after treatment with anthelmintic drugs. These fluorescent markers were efficient to stain larvae treated with ivermectin and albendazole sulfoxide. We observed that densitometric values were proportional to the concentration of dead larvae stained with both markers. Furthermore, data on motility test presented an inverse correlation with fluorimetric data when ivermectin was used. Our results showed that lower concentrations of drugs were effective to interfere in the processes of cellular transport while higher drugs concentrations were necessary in order to result in any damage to cell integrity. The methodology described in this work might be useful for studies that aim to evaluate the viability of nematodes, particularly for testing of new anthelminthic compounds using an easy, economic, reproducible, and no time-consuming technique.

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Analysis of viability using fluorimetric markers. (a) Microplate with serial dilution of C. elegans L3 (first point 2,000 to 62). Rows A–D dead larvae; E–H represent viable (control) larvae. Correlation between the number of dead larvae with 50% methanol and the fluorescence intensity of the pixels with Sytox (b) and propidium Iodide (c). Comparison of densitometry (number of pixels) between Sytox and propidium iodide markers (d). Significant differences (P < 0.05) were only detected when 1000 and 500 larvae were tested.
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fig2: Analysis of viability using fluorimetric markers. (a) Microplate with serial dilution of C. elegans L3 (first point 2,000 to 62). Rows A–D dead larvae; E–H represent viable (control) larvae. Correlation between the number of dead larvae with 50% methanol and the fluorescence intensity of the pixels with Sytox (b) and propidium Iodide (c). Comparison of densitometry (number of pixels) between Sytox and propidium iodide markers (d). Significant differences (P < 0.05) were only detected when 1000 and 500 larvae were tested.

Mentions: Both Sytox and propidium iodide were effective for staining larvae of C. elegans previously killed by treatment with 50% methanol (Figure 1), presenting a clear differentiation to viable larvae, which did not present any fluorescence (Supplementary Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/879263). When suspension of dead larvae was serially diluted and stained with both fluorescent markers (Figure 2(a)), the observed densitometric values were proportional to the concentration of dead larvae stained with Sytox (Figure 2(b)) and propidium iodide (Figure 2(c)). The comparison of the densitometric values obtained by staining with Sytox and propidium iodide demonstrates that significant differences were observed only at high number of larvae (500 and 1000 larvae) (P < 0.05) (Figure 2(d)).


A new methodology for evaluation of nematode viability.

Ferreira SR, Mendes TA, Bueno LL, de Araújo JV, Bartholomeu DC, Fujiwara RT - Biomed Res Int (2015)

Analysis of viability using fluorimetric markers. (a) Microplate with serial dilution of C. elegans L3 (first point 2,000 to 62). Rows A–D dead larvae; E–H represent viable (control) larvae. Correlation between the number of dead larvae with 50% methanol and the fluorescence intensity of the pixels with Sytox (b) and propidium Iodide (c). Comparison of densitometry (number of pixels) between Sytox and propidium iodide markers (d). Significant differences (P < 0.05) were only detected when 1000 and 500 larvae were tested.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383492&req=5

fig2: Analysis of viability using fluorimetric markers. (a) Microplate with serial dilution of C. elegans L3 (first point 2,000 to 62). Rows A–D dead larvae; E–H represent viable (control) larvae. Correlation between the number of dead larvae with 50% methanol and the fluorescence intensity of the pixels with Sytox (b) and propidium Iodide (c). Comparison of densitometry (number of pixels) between Sytox and propidium iodide markers (d). Significant differences (P < 0.05) were only detected when 1000 and 500 larvae were tested.
Mentions: Both Sytox and propidium iodide were effective for staining larvae of C. elegans previously killed by treatment with 50% methanol (Figure 1), presenting a clear differentiation to viable larvae, which did not present any fluorescence (Supplementary Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/879263). When suspension of dead larvae was serially diluted and stained with both fluorescent markers (Figure 2(a)), the observed densitometric values were proportional to the concentration of dead larvae stained with Sytox (Figure 2(b)) and propidium iodide (Figure 2(c)). The comparison of the densitometric values obtained by staining with Sytox and propidium iodide demonstrates that significant differences were observed only at high number of larvae (500 and 1000 larvae) (P < 0.05) (Figure 2(d)).

Bottom Line: Furthermore, data on motility test presented an inverse correlation with fluorimetric data when ivermectin was used.Our results showed that lower concentrations of drugs were effective to interfere in the processes of cellular transport while higher drugs concentrations were necessary in order to result in any damage to cell integrity.The methodology described in this work might be useful for studies that aim to evaluate the viability of nematodes, particularly for testing of new anthelminthic compounds using an easy, economic, reproducible, and no time-consuming technique.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil.

ABSTRACT
Nematodes infections are responsible for debilitating conditions and economic losses in domestic animals as well as livestock and are considered an important public health problem due to the high prevalence in humans. The nematode resistance for drugs has been reported for livestock, highlighting the importance for development of new anthelmintic compounds. The aim of the current study was to apply and compare fluorimetric techniques using Sytox and propidium iodide for evaluating the viability of C. elegans larvae after treatment with anthelmintic drugs. These fluorescent markers were efficient to stain larvae treated with ivermectin and albendazole sulfoxide. We observed that densitometric values were proportional to the concentration of dead larvae stained with both markers. Furthermore, data on motility test presented an inverse correlation with fluorimetric data when ivermectin was used. Our results showed that lower concentrations of drugs were effective to interfere in the processes of cellular transport while higher drugs concentrations were necessary in order to result in any damage to cell integrity. The methodology described in this work might be useful for studies that aim to evaluate the viability of nematodes, particularly for testing of new anthelminthic compounds using an easy, economic, reproducible, and no time-consuming technique.

Show MeSH
Related in: MedlinePlus