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Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells.

Osibote E, Noah N, Sadik O, McGee D, Ogunlesi M - Reprod. Biol. Endocrinol. (2011)

Bottom Line: All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm.This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm.Overall, the DOX results compared well with the conventional methods of checking proliferation of cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Center for Advanced Sensors & Environmental Monitoring, State University of New York at Binghamton, NY 13902-6000, USA.

ABSTRACT

Background: We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells.

Methods: The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96) and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence spectroscopy.

Results: This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%). DOX-96 showed a lower limit of detection (1.25 × 10(4) cells/ml); whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4) cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm.

Conclusions: Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring.

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MTT results for the residual activities of the fractions that are active (a)-Hexane fraction, (b) Butanol fraction, (c) Combined hexane fractions. Other conditions as in Figure 3.
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Figure 4: MTT results for the residual activities of the fractions that are active (a)-Hexane fraction, (b) Butanol fraction, (c) Combined hexane fractions. Other conditions as in Figure 3.

Mentions: At lower concentrations (10, 15, 20, 25 and 30 ppm) of the spiked extracts, the proliferation effect became more noticeable. The residual activities obtained for hexane fraction were 110-120% at 24 hrs while for the butanol fraction all the concentrations gave values between 105-110%. This observation might support the hypothesis that the plant extract contains active ingredients that will induce enhanced proliferation in the short-term, which perhaps will decrease or even result in toxicity upon prolonged exposure. Combining the hexane and butanol fractions, the effect was found to be more noticeable around 10 ppm concentrations with residual activity between 105-120% over that of control (Figure 4a-c). The proliferation effect was found to gradually increase from 1 ppm to 10 ppm and dropped drastically at 15 ppm for the hexane fraction; similar effect was recorded for the butanol fraction (Figure 4a-b). It should be noted that the extracts are most likely to give the proliferation effects at stages 8-14 during which degeneration of the cells are more noticeable and spermatogenesis progresses (Figure 1). The pattern of germ cell degeneration in the normal rat seminiferous epithelium is significantly altered when the availability of testosterone is reduced or completely withdrawn when germ cells at stage 7 are the first to degenerate and do so in large numbers compared with all other stages. These findings suggest that stage 7 in particular acutely depends upon an adequate supply of testosterone [11]


Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells.

Osibote E, Noah N, Sadik O, McGee D, Ogunlesi M - Reprod. Biol. Endocrinol. (2011)

MTT results for the residual activities of the fractions that are active (a)-Hexane fraction, (b) Butanol fraction, (c) Combined hexane fractions. Other conditions as in Figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117771&req=5

Figure 4: MTT results for the residual activities of the fractions that are active (a)-Hexane fraction, (b) Butanol fraction, (c) Combined hexane fractions. Other conditions as in Figure 3.
Mentions: At lower concentrations (10, 15, 20, 25 and 30 ppm) of the spiked extracts, the proliferation effect became more noticeable. The residual activities obtained for hexane fraction were 110-120% at 24 hrs while for the butanol fraction all the concentrations gave values between 105-110%. This observation might support the hypothesis that the plant extract contains active ingredients that will induce enhanced proliferation in the short-term, which perhaps will decrease or even result in toxicity upon prolonged exposure. Combining the hexane and butanol fractions, the effect was found to be more noticeable around 10 ppm concentrations with residual activity between 105-120% over that of control (Figure 4a-c). The proliferation effect was found to gradually increase from 1 ppm to 10 ppm and dropped drastically at 15 ppm for the hexane fraction; similar effect was recorded for the butanol fraction (Figure 4a-b). It should be noted that the extracts are most likely to give the proliferation effects at stages 8-14 during which degeneration of the cells are more noticeable and spermatogenesis progresses (Figure 1). The pattern of germ cell degeneration in the normal rat seminiferous epithelium is significantly altered when the availability of testosterone is reduced or completely withdrawn when germ cells at stage 7 are the first to degenerate and do so in large numbers compared with all other stages. These findings suggest that stage 7 in particular acutely depends upon an adequate supply of testosterone [11]

Bottom Line: All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm.This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm.Overall, the DOX results compared well with the conventional methods of checking proliferation of cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Center for Advanced Sensors & Environmental Monitoring, State University of New York at Binghamton, NY 13902-6000, USA.

ABSTRACT

Background: We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells.

Methods: The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96) and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence spectroscopy.

Results: This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%). DOX-96 showed a lower limit of detection (1.25 × 10(4) cells/ml); whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4) cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm.

Conclusions: Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring.

Show MeSH