How the Seahorse Mito Stress Test is Used in Cancer Spheroids

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How the Seahorse Mito Stress Test is Used in Cancer Spheroids

The Seahorse Mito Stress Test enables the imaging of mitochondrial function and their toxicity on cell metabolism. The test is highly specific and can determine the role of mitochondrial dysfunction in oxidative stress, respiration, and membrane potential. It can be performed on cancer spheroids and other cell types. In this article, we will discuss how this technique is used in cancer spheroids and discuss the implications of its findings.

Single Spheroid Protocol

The Seahorse XF Cell Mito Stress Test Kit enables quantification of mitochondrial function in 9-month-old cerebral organoids. The test measures the effects of perturbations on mitochondrial respiration. The cells were cultured in a microplate containing a 10 mg/mL laminin coating. The cells were then cut into three sections. The Seahorse Wave desktop software was used to analyze the results.

After treatment, 180 uL of MAS were replaced with a mixture of 10 mM pyruvate, 1 mM malate, and 4 mM ADP. To assess the ECAR, the lower asymptote of OCR was set at 0% and the upper asymptote was set at 100%, which corresponds to the maximal ECAR induction. The variations in all the assays were calculated as standard deviations and the curves fitted with a nonlinear regression four-parameter Hill model. Sucrose was injected sequentially.

Alternative normalization strategies

The Seahorse Mito Stress Test (SHORE) measures the OCR of several breast cancer cell lines. MCF7 and MDA-MB-231 are known to have high OCR. The authors used a normalization strategy that accounts for the different basal OCR levels of these cell lines. The following example shows how this normalization strategy works:

ECAR and protein normalization have their own advantages and limitations. While protein normalization does not distinguish glycolytic function, it can help researchers compare the two cell lines. Live nuclear dyes emit a brighter fluorescence signal, but they have no cytotoxic effect. Both methods are capable of normalizing Seahorse(tm) XF data. They can be used in the same workflow.

ECAR and OCR measurements can be calculated using multiple spheroids. The Single Spheroid Protocol reduces CVs by a significant margin. The Multiple Spheroid Protocol results in the highest variability, but the Single Spheroid Protocol produces consistent Mito Stress profiles. A common mistake in interpreting the data is to use one method over the other. In this case, you should choose the strategy that best fits your study.


The data derived from the Seahorse XF mito stress test are interpreted using the metabolomic phenotype. These results are presented as mean + standard error of the mean (SEM). The Seahorse XF24 is a multi-parameter extracellular flux analyzer. It measures oxygen consumption rates, glycolytic activity, and extracellular acidification.

In addition to evaluating the OCR, XF analysis can also determine cellular bioenergetics. This technique measures basal and ATP-associated oxygen consumption rates in PBMCs. The data is useful in determining optimal cell numbers, as the optimum number of cells enables the study of various cellular parameters. Using the XF assay, scientists can determine the optimal cell density for each platform.

The XF cell stress test is a powerful, customizable solution for assessing mitochondrial function. By measuring several parameters in one assay, the Seahorse XF Mito Stress Test can help researchers determine whether a drug inhibits mitochondrial function. It also allows them to identify functional differences between various cell types. The XF mito stress test kit measures maximal and non-mitochondrial respiration, and removes the need for solvents in the process.

Application to cancer spheroids

To investigate the mitochondrial respiratory capacity of cancer spheroids, we developed an assay that probes the metabolic parameters of mitochondrial oxidative phosphorylation in 3D cultured cells. We measured OCR from spheroids seeded with different cell densities. As the spheroid size increased, OCR linearly correlated with cell volume. The maximum OCR was measured in MCF-7 spheroids, while the lowest values were observed for SK-OV-3 and A549 spheroids.

We studied the mitochondrial energy metabolism of single 3D spheroids using a XFe96 XF Analyzer. We also used cell-repellent technologies to cultivate cellular spheroids quickly. Finally, we examined the effects of various therapeutic modalities on the spheroid’s metabolic network. While we cannot predict what treatments will work in each patient, our assay has the potential to identify promising drug targets.

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