Hello, I use the protocol to detect GSH and GSSG concenteration...

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LIAO liping
Oct 17, 2019
protocol Protocol: Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate
Hello, I use the protocol to detect GSH and GSSG concenteration in HEK293T cell line which is treated with drug and the same DMSO as control ,I do have some interesting problems: first, When I do the standard cuvre ,I realized that the signal of GSH is decrease with time increase but the Read more Read less more less
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LIAO liping Answered Oct 22, 2019

Shanghai Insitute of Materia Medica

Hi Vikas Singh

Thank you for your reply , And I have another question , I use this method to detect HEK293T cell lines GSH/GSSG ,I get each concentration as the standard curve, But the ratio of GSH/GSSG is about 5(three independent assays),but most paper reported that GSH/GSSG is about 100,much higher than I detect ,I didn't know whether there some thing I missed, I degassed all my buffer before use and use fresh cell lysate to detect GSH and GSSG, What's your opinion, So thank you for your time .
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Vikas Singh Author Answered Oct 17, 2019

Immunotoxicology Laboratory, Food Drug and Chemical Toxicology Group and Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), 31-Vishvigyan Bhawan, India

Hi Liao liping

First query is about getting lower fluorescence values in GSH standard with time than GSSG. I think this may be due to the oxidation of GSH into GSSG with time. My suggestion to you is to every time make a fresh GSH and GSSG standards for your experiments. Secondly it is not required to take readings till 24 hrs you can consider 10 or 15 mins readings for your standard plot as this much time is generally sufficient for reaction to reach its sarturation.

It is possible that you might get increase in fluorescence values in blank wells (this is due to OPT) but that is not much of a concern because during your calculation part you should substract the blank value from known concentrations of GSH and GSSG.

I don't think preincubation of GSSG with NaOH solution is right way of performing the experiments as GSSG hydrolysis happens very quickly with NaOH so there is no requirement of preincubation.

I also noticed that you are shaking and centrifuging your samples after OPT addition. This is not required you just have to use multichannel pippette for mixing samples which also reduces your time of processing before capturing fluorescence.

Finally readings you are getting are different from ours this may be due to different gain settings in plate reader (Fluorescence values change if gain settings are altered).

I hope i am able to answer your queries clearly.

Best of luck for your experiments.
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