Mitochondrial Metabolic Assay using Seahorse™ - Newcells

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The most advanced near-physiological high throughput kidney proximal tubule cells (PTC) model to investigate drug transport modalities in vitro.

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Mitochondrial Metabolic Assay using Seahorse™

Kidney Tubule Cells contain a large number of mitochondria to handle drugs and excrete xenobiotics. Using the Seahorse mitochondrial metabolic assay we can evaluate mitochondrial health, providing a good indicator of overall cell heath and evaluation of drug safety.

Mitochondrial Health in Proximal Tubule Cells

  • The kidney removes waste and toxins from the body
  • Proximal Tubule Cells (PTCs) are responsible for the reabsorption of solutes and the excretion of xenobiotics.
  • PTCs express a large number of transporters and contain a large number of mitochondria.
  • Mitochondria generate the energy (ATP) that keeps the cells healthy and functional.
  • Some drugs affect mitochondrial function causing damage to PTCs and the loss of renal function.
  • For new drugs, it is recommended to use the Seahorse mitochondrial metabolic assay to monitor mitochondrial health in PTCs and to avoid drugs inducing cell damage.

Coloured Transmission electron microscopy (TEM) of a kidney tubule cells. Basal infoldings are labelled in red colour, mitochondria in green, and lysosomes in blue

Three Assays to Check Mitochondrial Health

Drugs can affect cell metabolism and mitochondrial function. New drugs require testing
in vitro to assess how they are handled by PTCs and whether they cause damage to the cell and affect mitochondria. Reliable models are needed to test the effect of new drugs on mitochondrial function.

Newcells provides these assays to monitor mitochondrial health in kidney PTCs:

  • Mitochondrial Function – Agilent Seahorse XF Assay
  • Mitochondrial Membrane Potential (MMP) – tetramethyl rhodamine methyl ester assay
  • Oxidative Stress – Measurement of reactive oxygen species (ROS) production

De-risk your drug discovery pipeline with aProximate™

aProximate™ – The most advanced near-physiological high throughput kidney proximal tubule cell (PTC) model.

  • Most advanced near-physiological PTC model
  • High throughput solution for industry
  • Expression of all key renal transporters
  • Outperforms competitor in vitro models
  • FDA approved kidney biomarkers
  • Enables mitochondrial health monitoring

1. Mitochondrial Function (using the Agilent Seahorse XF Assay)

Normal cells rely on mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP as a source of energy.

The Seahorse XF assay measures the oxygen consumption rate (OCR) and pH or extracellular acidification rate (ECAR) in the media surrounding living cells. It is a fast, real-time, label-free, sensitive and precise measurement of cellular metabolism that correlates with mitochondrial function.

Mitochondrial bioenergetics explained. Basal OCR drops when ATP production is stopped by the addition of ATPase inhibitor Oligomycin, leaving proton leak and non–mitochondrial OCR . Addition of the uncoupler FCCP restores maximal respiration, whilst addition of inhibitor Antimycin A (AMA) blocks mitochondrial oxygen consumption.

Effect of Cisplatin on Mitochondrial Function

Cisplatin, a cytotoxic anti-cancer drug, negatively affects mitochondrial function and cell metabolism.

The Seahorse XF assay detects changes in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) simultaneously, without disrupting the cells upon addition of cisplatin.

Cisplatin is shown to inhibit OCR and ECAR in aProximate™ PTCs in vitro model.

Dose-dependent inhibition of OCR and ECAR in aProximate™ PTCs by the cytotoxic drug cisplatin

State-of-the-art 2D and 3D cell-based assays

2. Mitochondrial Membrane Potential (MMP) Assay

Mitochondrial function can be assessed by monitoring changes in mitochondrial membrane potential (MMP). Preservation of mitochondrial membrane potential is an indicator of cell health. MMP assay measures tetra-methyl rhodamine methyl ester accumulation in the mitochondria and it is an indicator of cell health.

MMP assay principle. In healthy cells the mitochondrial membrane potential indicator (MPI) dye accumulates in mitochondria as aggregates (red fluorescence can be detected). When mitochondrial potential collapses MPI dye is mainly distributed in the cytoplasm as monomers (green fluorescence).

3. Oxidative Stress Assessment

Oxidative stress is a caused by the imbalance between production and accumulation of reactive oxygen species (ROS) in the cells. ROS are produced as a result of mitochondrial damage by some drugs and xenobiotics. ROS production, measured using a fluorescent probe, is an indication of cell health.

Newcells provides oxidative stress assay in aProximate™ PTCs.

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