Oligo/FCCP/Rot/AA drug recommended conc for human melanoma cell lines (A375 and SKMEL5)

Our lab is trying to run Seahorse XFe96 Cell Mito Stress Test on human melanoma cell lines. I was wondering what would be the recommended concentrations for Oligomycin, FCCP, Rotenone and Antimycin in this particular cell line. We usually prepare the stock solutions ourselves

  • Hi Christian,

    Thank you for contacting Cell Analysis Technical Support. I can see that you are already in communication with my colleague, Raji, so I will let you continue your conversation via email.

    Thank you,


    Courtney Nadeau Watts

    Technical Support Scientist/Remote Engineer

    Cell Analysis Products

  • For Cell Mito stress test, Seahorse Cell Mito Stress Test Kit, the recommended working concentrations are 1.5 µM for Oligomycin and 0.5 µM for Rot/AA. The FCCP concentration needs to be optimized (0.5 µM-2 µM). The optimal final compound concentration for achieving maximal effect is cell line dependent, and may be affected by assay medium types. Therefore, it is recommended that, for each new cell line or assay medium, a titration experiment for the compounds is performed. This is especially important with FCCP, as the titration curve tends to be quite sharp, and too much FCCP can actually diminish responses in OCR. For details refer to Seahorse XF Cell Mito Stress Test Kit User Guide (agilent.com). The assays are validated with Agilent reagents, thus we cannot provide any guaranteed concentrations for in-house reagents. You may need to optimize further.

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