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Oligomycin is not working

Chanyoung

Hello, I am trying to measure OCR with FHs74 Int cell line (ATCC) and optimizing the concentrations of Oligomycin, FCCP, and Rotenone/antimycin A.

I confirmed that FCCP works at 1 μM and 5 μM, and Rotenone/antimycin A works at 0.5 μM.

However, Oligomycin does not work at 1.5, 2, 2.5, or 3 μM (the OCR graph line does not decrease after Oligomycin injection).

I don't know how to solve this issue, so I would like to get some advice.

Note: This is an untreated, normal state of FHs74 Int cell line.

I look forward to hearing from you.

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  • 0

    Hi Chanyoung,

    Thank you for contacting Cell Analysis Technical Support. We recommend using 1.5uM oligo concentration for most cell lines (when using our kits). Are you using an Agilent kit? If you would like, I can take a look at your data. You can send it to cellanalysis.support@agilent.com. 

    Thank you!


    Courtney 

    Courtney Nadeau Watts

    Technical Support Scientist/Remote Engineer

    Cell Analysis Products

  • 0 in reply to Courtney Watts

    Hi, Courtney Watts

    I have just sent my experimental data via email with the subject line "Data for the question 'Oligomycin is not working.'"

    Instead of using a kit, I used the product purchased from Sigma for Oligomycin.

    I have another question.
    I observed another researcher adding 1% BSA (free fatty acid) to the analysis buffer for OCR measurement experiments. However, the Agilent protocol did not include BSA. I am curious about how this might affect the experiment.

    Thank you for your time and consideration.

  • 0 in reply to Chanyoung

    Hi Chanyoung,

    I will look out for your results files to see if I can see anything technically wrong in your data. However, we cannot troubleshoot reagents from another vendor as we cannot speak to the quality of the products.  

    Regarding BSA: some cells/applications do need BSA during the assay (example - XF Palmitate Oxidation Stress Test - Advanced Assay where BSA is needed to keep palmitate in solution). If this is necessary, BSA containing solutions should not be loaded into the ports as this will cause issues with injections. Additionally, if there is BSA present, FCCP will need to be re-titrated as BSA will bind to FCCP, reducing its efficacy. 

    Thank you,


    Courtney

  • 0 in reply to Courtney Watts

    Hi, Courtney Watts

    Thank you for your kind response.

    I have sent the necessary data again via email.

    Please check it.

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