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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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Fluorescence assay results for the residual activities of the active fractions at smaller concentration intervals. (a) Hexane fraction, (b) Butanol fraction, (c) combined/mixed fractions of hexane and butanol. Optimum cell culture conditions: 5-10% CO2, 90% humidity and a constant temperature of 37°C. Other conditions are as in Figure 5.
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Figure 6: Fluorescence assay results for the residual activities of the active fractions at smaller concentration intervals. (a) Hexane fraction, (b) Butanol fraction, (c) combined/mixed fractions of hexane and butanol. Optimum cell culture conditions: 5-10% CO2, 90% humidity and a constant temperature of 37°C. Other conditions are as in Figure 5.

Mentions: At lower concentrations: 2 - 15 ppm (Figure 6a), the hexane fraction showed proliferation effect at 24 hrs incubation with values ranging between 120 and 180% of the control, and no effect was recorded 48 and 72 hrs respectively. The butanol fraction (Figure 6b) also showed more effect at 24 hrs than 48/72 hrs with values raging from 105-150%. Combining the two fractions (Figure 6c) the proliferation effect was also higher at 24 hrs incubation with values between 110-120%. 10 - 20 ppm produced the highest values for at 24 and 48 hrs incubation. However, a reduction effect was noticed as the concentration increased after 72 hrs and no proliferation effect was recorded [24]. Overall, the most significant proliferation effect was recorded at 10 ppm spiked extract concentration. Based on these observations, subsequent experiments were carried out at lower concentrations in order to observe possible trends at smaller increases (15-30 ppm) in extract concentrations.


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)

Fluorescence assay results for the residual activities of the active fractions at smaller concentration intervals. (a) Hexane fraction, (b) Butanol fraction, (c) combined/mixed fractions of hexane and butanol. Optimum cell culture conditions: 5-10% CO2, 90% humidity and a constant temperature of 37°C. Other conditions are as in Figure 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Fluorescence assay results for the residual activities of the active fractions at smaller concentration intervals. (a) Hexane fraction, (b) Butanol fraction, (c) combined/mixed fractions of hexane and butanol. Optimum cell culture conditions: 5-10% CO2, 90% humidity and a constant temperature of 37°C. Other conditions are as in Figure 5.
Mentions: At lower concentrations: 2 - 15 ppm (Figure 6a), the hexane fraction showed proliferation effect at 24 hrs incubation with values ranging between 120 and 180% of the control, and no effect was recorded 48 and 72 hrs respectively. The butanol fraction (Figure 6b) also showed more effect at 24 hrs than 48/72 hrs with values raging from 105-150%. Combining the two fractions (Figure 6c) the proliferation effect was also higher at 24 hrs incubation with values between 110-120%. 10 - 20 ppm produced the highest values for at 24 and 48 hrs incubation. However, a reduction effect was noticed as the concentration increased after 72 hrs and no proliferation effect was recorded [24]. Overall, the most significant proliferation effect was recorded at 10 ppm spiked extract concentration. Based on these observations, subsequent experiments were carried out at lower concentrations in order to observe possible trends at smaller increases (15-30 ppm) in extract concentrations.

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