This standard curve was generated using the LEGENDplex™ Human Adipokine Panel. A standard curve must be run with each assay.

Measuring the metabolic response

Cytokines and hormones help regulate the body’s metabolic response. Altered levels of these proteins have been associated with a variety of metabolic diseases and inflammatory autoimmune conditions. This makes it pivotal to accurately measure adipose tissue proteins to gain a better understanding of disease progression and immune responses.

LEGENDplex™ panels use antibody-coated beads to multiplex over a dozen targets and quantify protein concentrations using a flow cytometer. Explore our pre-optimized metabolism panels below or mix and match specific analytes to fit your experiments.

Human LEGENDplex™ panels
Adipokine Panel
Apolipoprotein (Apo) Panel
Bone Metabolism Panel V02
Diabesity Panel
Metabolic Panel 1
TNFSF Family Panel 1

(Left) Total lysates (15 µg protein) from HeLa (Human), Raw264.7 (Mouse), UMR106 (Rat) and CHO (Hamster) were resolved by electrophoresis (4-20% Tris-glycine gel), transferred to nitrocellulose, and probed with 1:1000 purified anti-Cytochrome C antibody. Proteins were visualized using chemiluminescence detection by incubation with HRP Goat anti-Mouse secondary antibody (1:3000 dilution). Non-specific band was marked with “*”. Direct-Blot™ HRP anti-β-actin was used as a loading control (1:8000 dilution).

(Right) HeLa cells were treated with 400 nM MitoSpy™ Green FM (green) for 30 minutes, fixed with 4% PFA for 15 minutes, permeabilized with 0.5% Triton X-100 for three minutes, and blocked with 5% FBS for 60 minutes. Then the cells were intracellularly stained with purified anti-Cytochrome c antibody overnight at 4°C followed by Alexa Fluor® 594 goat anti-mouse IgG (red) for one hour at room temperature (1:250 dilution, 2 µg/mL). Nuclei were counterstained with DAPI (blue). The image was captured with a 60X objective. The image was captured with a 60X objective using KEYENCE BZ-X700 fluorescence microscope. Exposure time (in seconds) is 1/20.

Antibodies abound

As industry leaders on antibody purification, conjugation, production, and quality control, we have generated dozens of useful clones for metabolic analysis. Our antibodies can be used for flow cytometry multicolor panels alongside optimized buffers and fixatives for sample preparation. We also provide antibodies for non-flow applications such as microscopy and western blotting.

Flow cytometry
Arginase-1, clone 14D2C43
CD98 (Human), clone 2B6/CD98.Rec
Cytochrome c, clone 6H2.B4
HIF1α (Human), clone 546-16
Nos2 (iNOS), clone W16030C

Additional applications
AMPK
Cytochrome c, clone 7H8.2C12
Hexokinase II
mTOR
SDHA
SDHB
Sortilin

Live cell fluorescence imaging of HeLa cells without (panel A) and with (panel B) carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment (5 µM for 30 min at 37°C), a potent mitochondrial decoupling agent. Then, cells were probed with JC-10 Mitochondrial Membrane Potential Kit. JC-10 aggregates can be visualized by red fluorescence, while JC-10 monomers which have diffused out of the mitochondria is visualized by green fluorescence. Nuclei were stained with Hoechst. Images were captured using a 40X objective. Scale bar: 15 µm

• Interrogate metabolic pathways at a single-cell level within a heterogeneous population
• Rapid – no fixation/permeabilization steps
• Does not require specialized equipment – detectable by flow cytometry or fluorescence microscopy
• Unlimited functional profiling (glycolysis, OxPhos, fatty acid oxidation, and other metabolic pathways).

Mitochondria spark metabolic pathways

Mitochondria are the powerhouses of the cell, using components of sugars, amino acids, and fatty acids to create ATP and GTP. In addition, mitochondria serve as a metabolic hub, breaking down complex nutrients for energy, creating biosynthetic precursors, maintaining redox homeostasis, and overseeing metabolic waste management. As it oversees these chemical reactions and ATP production, a membrane potential is created along the mitochondrial membrane by the electron/ion transport chain. Mitochondrial dysfunction or loss of membrane potential is often a sign of distress in a cell and can lead to apoptosis.

Many chemical probes, such as MitoSpy Orange, Red, and NIR, can recognize mitochondria membrane potential and bind to the organelle. These MitoSpy™ formats can indicate cell health as well as localization. MitoSpy™ Green's attraction to the mitochondria is not based on membrane potential, allowing it to measure mitochondrial mass of individual cells in flow cytometry. Our chemical probes assess mitochondrial membrane potential and reactive oxygen species levels, providing keen insight on cell health.

Our JC-10 Mitochondrial Membrane Potential Kit qualitatively monitors and visualizes apoptosis by fluorescence microscopy, flow cytometry, or plate reader analysis.

• View ATP Red standalone components - ATP Red, 2-Deoxy-D-Glucose, Oligomycin