Oxidative Stress Assay in Cells

This assay was performed as described [87 (link)]. Briefly, asynchronous cells in log phase were incubated with 10μg/ml dihydrodichloro-fluorescein diacetate (D6883 (Sigma) from 2.5mg/ml stock prepared in 100% ethanol) for 2 hours at 30°C in the dark with gentle mixing. After incubation, cells were spotted on polylysine-coated slides and immediately visualized using a Zeiss Axioskop 40 microscope equipped with a green filter set (Zeiss filter set #38) and a CCD camera for image acquisition. >100 cells were counted for each sample.

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Vijayraghavan S., Tsai F.L, & Schwacha A. (2016). A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M. PLoS Genetics, 12(8), e1006277.

Publication 2016

D6883 Axioskop 40 Zeiss filter set #38

Assay Cells Ethanol Fluorescein diacetate Microscope Polylysine

Corresponding Organization : University of Pittsburgh

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Variable analysis

independent variables

Dihydrodichloro-fluorescein diacetate (D6883 (Sigma) from 2.5mg/ml stock prepared in 100% ethanol)

dependent variables

Fluorescence intensity (visualized using a Zeiss Axioskop 40 microscope equipped with a green filter set (Zeiss filter set #38) and a CCD camera for image acquisition)

control variables

Asynchronous cells in log phase
Incubation time (2 hours)
Incubation temperature (30°C)
Incubation conditions (in the dark with gentle mixing)
Slide preparation (polylysine-coated slides)

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