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High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Performance of the high-content oxidative stress assay with human embryonic stem cell (hESC)-differentiated astrocytes in a screen against the NPC chemical library. A total of eight 1,536-well assay plates with hESC-differentiated astrocytes were screened against 2,816 compounds in titration in the NPC chemical library. Although the S/B was low for the screen (A, D), the variability was also very low for most parameters (C), which yields decent Z′ values for many parameters (B, E). (F): The positive control, terfenadine, was tested in titration on each assay plate as a measure to assess the performance and reproducibility of the assay in the screen. Shown are nuclear intensity concentration response curves for the terfenadine titration on each of the eight assay plates screened. Abbreviations: %CV, coefficient of variation; AC50, concentration of compound that gives half-maximal response; DMSO, dimethyl sulfoxide; FDA A and FDA B, subsets of the NPC compount library; NPC, NIH Chemical Genomics Center Pharmaceutical Collection; RFU, relative fluorescence unit; S/B, signal/basal.
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Figure 3: Performance of the high-content oxidative stress assay with human embryonic stem cell (hESC)-differentiated astrocytes in a screen against the NPC chemical library. A total of eight 1,536-well assay plates with hESC-differentiated astrocytes were screened against 2,816 compounds in titration in the NPC chemical library. Although the S/B was low for the screen (A, D), the variability was also very low for most parameters (C), which yields decent Z′ values for many parameters (B, E). (F): The positive control, terfenadine, was tested in titration on each assay plate as a measure to assess the performance and reproducibility of the assay in the screen. Shown are nuclear intensity concentration response curves for the terfenadine titration on each of the eight assay plates screened. Abbreviations: %CV, coefficient of variation; AC50, concentration of compound that gives half-maximal response; DMSO, dimethyl sulfoxide; FDA A and FDA B, subsets of the NPC compount library; NPC, NIH Chemical Genomics Center Pharmaceutical Collection; RFU, relative fluorescence unit; S/B, signal/basal.

Mentions: Once the high-content oxidative stress assay using hESC-differentiated astrocytes was optimized, we screened a set of 4,096 compounds that consisted of bioactive tool compounds and approved drugs. The performance for these two compound screens was similar (Fig. 3; supplemental online Fig. 7). Although the S/B ratio comparing the DMSO/vehicle-treated cells to H2O2-treated cells was low for most nuclear parameters (Fig. 3A; supplemental online Fig. 7A), the well-to-well variability, as measured by the percent coefficient of variation (%CV) was also extremely low—approximately 1% for most parameters in both screens (Fig. 3C; supplemental online Fig. 7C), resulting in Z′ values of approximately 0.5 for some nuclear parameters (Fig. 3B; supplemental online Fig. 7B). The supplemental online data defines Z′ value. The low S/B ratio when comparing the DMSO group with the H2O2-treated group was partially the result of using assay conditions in which only a fraction of the astrocytes underwent cell death processes. A noticeably larger S/B ratio was seen when astrocytes were treated with 100 µM terfenadine for 24 hours (Figs. 2A [inset], 3D; supplemental online Fig. 7D). This generated Z′ values greater than 0.5 for many of the nuclear parameters in both screens (Fig. 3E; supplemental online Fig. 7E).


High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress
Performance of the high-content oxidative stress assay with human embryonic stem cell (hESC)-differentiated astrocytes in a screen against the NPC chemical library. A total of eight 1,536-well assay plates with hESC-differentiated astrocytes were screened against 2,816 compounds in titration in the NPC chemical library. Although the S/B was low for the screen (A, D), the variability was also very low for most parameters (C), which yields decent Z′ values for many parameters (B, E). (F): The positive control, terfenadine, was tested in titration on each assay plate as a measure to assess the performance and reproducibility of the assay in the screen. Shown are nuclear intensity concentration response curves for the terfenadine titration on each of the eight assay plates screened. Abbreviations: %CV, coefficient of variation; AC50, concentration of compound that gives half-maximal response; DMSO, dimethyl sulfoxide; FDA A and FDA B, subsets of the NPC compount library; NPC, NIH Chemical Genomics Center Pharmaceutical Collection; RFU, relative fluorescence unit; S/B, signal/basal.
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Related In: Results  -  Collection

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Figure 3: Performance of the high-content oxidative stress assay with human embryonic stem cell (hESC)-differentiated astrocytes in a screen against the NPC chemical library. A total of eight 1,536-well assay plates with hESC-differentiated astrocytes were screened against 2,816 compounds in titration in the NPC chemical library. Although the S/B was low for the screen (A, D), the variability was also very low for most parameters (C), which yields decent Z′ values for many parameters (B, E). (F): The positive control, terfenadine, was tested in titration on each assay plate as a measure to assess the performance and reproducibility of the assay in the screen. Shown are nuclear intensity concentration response curves for the terfenadine titration on each of the eight assay plates screened. Abbreviations: %CV, coefficient of variation; AC50, concentration of compound that gives half-maximal response; DMSO, dimethyl sulfoxide; FDA A and FDA B, subsets of the NPC compount library; NPC, NIH Chemical Genomics Center Pharmaceutical Collection; RFU, relative fluorescence unit; S/B, signal/basal.
Mentions: Once the high-content oxidative stress assay using hESC-differentiated astrocytes was optimized, we screened a set of 4,096 compounds that consisted of bioactive tool compounds and approved drugs. The performance for these two compound screens was similar (Fig. 3; supplemental online Fig. 7). Although the S/B ratio comparing the DMSO/vehicle-treated cells to H2O2-treated cells was low for most nuclear parameters (Fig. 3A; supplemental online Fig. 7A), the well-to-well variability, as measured by the percent coefficient of variation (%CV) was also extremely low—approximately 1% for most parameters in both screens (Fig. 3C; supplemental online Fig. 7C), resulting in Z′ values of approximately 0.5 for some nuclear parameters (Fig. 3B; supplemental online Fig. 7B). The supplemental online data defines Z′ value. The low S/B ratio when comparing the DMSO group with the H2O2-treated group was partially the result of using assay conditions in which only a fraction of the astrocytes underwent cell death processes. A noticeably larger S/B ratio was seen when astrocytes were treated with 100 µM terfenadine for 24 hours (Figs. 2A [inset], 3D; supplemental online Fig. 7D). This generated Z′ values greater than 0.5 for many of the nuclear parameters in both screens (Fig. 3E; supplemental online Fig. 7E).

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.