Study type: In vitro · Status: Evidence verified by machine against declared source
Quantitative real-time imaging of glutathione
Nature communications · 2017
Study scale: To ensure data quality and reproducibility, at least two biological replicates with >6 cells were analysed for each imaging experiment, and all the imaging experiments were repeated at least twice with one set of representative data shown in each figure.
Abstract only: Open source record
Product or molecular entity relationships
- Glutathione: Exact entity relationship. Legacy citation custody associates this source with the catalog record; no product-relevance conclusion is implied.
Public plain-language summary
The published text is limited to the declared source and preserves reported uncertainty. It does not establish efficacy, safety, suitability, or evidence strength.
Research question
We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.
Study design
In accordance with previous studies2829, we administered a ferroptosis inducer, erastin, to HT1080 cell culture and observed decrease in GSH levels by flow cytometry of RT signals (Fig. 7a,b).
Participants or experimental system
Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown.
Study scale
To ensure data quality and reproducibility, at least two biological replicates with >6 cells were analysed for each imaging experiment, and all the imaging experiments were repeated at least twice with one set of representative data shown in each figure.
Intervention or exposure
To assess whether RT probe binds to any proteins or reacts with free cysteine residues in some proteins that may interfere with the final ratio readouts, we incubated RT with cell lysate, which was dialyzed using a 3 K cut-off membrane to remove all small molecules including GSH, and observed no significant changes in fluorescent signal ratios even at the highest lysate concentration shown (protein concentration is 36 mg ml−1 based on a BCA assay), suggesting that there is no non-specific interactions or reactions between cellular proteins and RT which could significantly change the ratio readouts (Fig. 2a).
Comparator
Cells were cultured under a controlled atmosphere (37 °C, 5% CO2).
Outcomes examined
Additionally, the intracellular GSH level changes measured using RT were confirmed by cell lysate-based liquid chromatography–mass spectrometry (LC–MS) measurements (Fig. 4d).
Key findings
Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis.
Limitations and uncertainty
Because inhomogeneous cellular distribution of reacted and unreacted probes may bias the ratiometric calculations, especially at the subcellular resolution, we opted for RT instead of RT-NH2 to achieve high-quality quantitative results in living cells.
Product relevance and evidence boundary
This record is a published study, held here with its citation and review status. It is not a statement that any catalog item is effective, safe, or suitable for any use, and nothing in it is a dose or a protocol.
Evidence verified by machine against declared source. Verification is limited to the declared source and review scope. It does not mean independent replication or establish efficacy, safety, or suitability.