I am working with fibroblasts and recently conducted a mito stress test using seahorse technology. Surprisingly, I observed unexpectedly low basal oxygen consumption rates (OCR). Furthermore, instead of the anticipated increase in OCR following the oligomycin pulse, I observed a decrease. I'm puzzled about the potential reasons behind this unexpected outcome. Could you provide any insights or suggestions to help me identify the problem?
Your first question is about low basal OCR. The first few questions that come to mind for troubleshooting this are:
- Do you have enough cells in each well? It looks like Agilent recommends 20×10^3 cells/well for human skin fibroblasts, but I don't know what your protocol says to do. If there aren't enough cells then you will be below the minimum detection limit for the oxygen probe. The output will then be a negative value because the machine is reading zero and then the value of the blanks is automatically subtracted from it.
- Is it plated evenly? The cells should settle to the bottom of the plate in a relatively even layer. Or are they adherent?
- Do you have enough blanks in your plate? How many depends on if you are using a 24 well or 96 well plate. I'd say 4 for a 24 well plate at at least 8 for a 96 well.
- Have you designated blank wells as "blanks" in the software? Because Wave software automatically subtracts blanks from other wells.
- Have you entered the correct total well volume in the "Run Assay" tab? This is the starting well volume plus the injection volumes.
- It looks like you have it set to "normalize off". Have you have normalized results to cell number separately? You can have the software do it.
Your second question is about oligomycin. Oligomycin inhibits complex V in mitochondria (ATP synthase) causing a decrease in the flow of electrons through the electron transport chain. This decreases ATP-linked respiration and results in decreased OCR. This allows you to calculate ATP production. So if you are seeing a decrease from basal after the injection of oligomycin then it is actually working correctly.
I recommend looking through these protocols to help with additional troubleshooting for your problem:
- Mishra P, Zhang T, Guo M, Chan D. Mitochondrial Respiratory Measurements in Patient-derived Fibroblasts. Bio Protoc. 2019 Dec 5;9(23):e3446. doi: 10.21769/BioProtoc.3446.
- Gu X, Ma Y, Liu Y, Wan Q. Measurement of mitochondrial respiration in adherent cells by Seahorse XF96 Cell Mito Stress Test. STAR Protoc. 2020 Dec 30;2(1):100245. doi: 10.1016/j.xpro.2020.100245.
- Also search for Agilent's "Report Generator User Guide"
I hope that helps.
- Melissa
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