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Glycolysis/Mitochondrial Membrane Potential Measurement Reagents

Table of Contents

Glycolysis and Oxidative Phosphorylation

Glycolysis and oxidative phosphorylation are the two main ATP-producing metabolic pathways within cells, closely linked via a common intermediate, pyruvate (Figure 1).1,2) Upon transportation into mitochondria, pyruvate is converted into acetyl CoA, which subsequently enters the citric acid (Krebs) cycle to facilitate oxidative phosphorylation. However, under conditions of enhanced glycolytic activity or impaired mitochondrial function, pyruvate is instead converted into L-lactate through the action of L-lactate dehydrogenase (LDH) and subsequently released into the extracellular space. Therefore, the amount of L-lactate produced serves as an indicator of the extent to which glycolysis is preferentially activated.3)

In contrast, oxidative phosphorylation occurs via the electron transport chain and ATP synthase, both of which are located in the inner mitochondrial membrane, the latter using the mitochondrial membrane potential generated by the former to efficiently generate ATP. Consequently, the mitochondrial membrane potential can be considered directly indicative of the activity of oxidative phosphorylation and the functional state of mitochondria.2)

As described above, enhanced glycolysis activity manifests as increased L-lactate production, while the status of oxidative phosphorylation can be assessed using mitochondrial membrane potential. Therefore, measuring both L-lactate production and mitochondrial membrane potential enables the balance between glycolysis and oxidative phosphorylation to be evaluated, providing an insight into the cellular energy metabolic profile.4)

Primary metabolic pathway in cells
Figure 1. Primary metabolic pathway in cells

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Glycolysis Assay Reagent

L-Lactate Assay Kit (Product No. L0507 enables the straightforward quantification of L-lactate both in cell culture supernatants and intracellularly. The L-lactate concentration in samples can be accurately determined using a calibration curve created with the supplied Lactate Standard Solution. The measurable L-lactate concentration range is 0.02 to 1.00 mM. Samples with a concentration exceeding 1.00 mM can be measured via dilution.

Product

L0507
L-Lactate Assay Kit

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Kit Components

  • Lactate Dye Mixture 1 Vial
  • Lactate Enzyme Mixture 200 µL
  • Lactate Cofactor Mixture 500 µL
  • Lactate Assay Buffer 8 mL
  • Lactate Standard Solution 200 µL

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Example of use 1 (Quantification of L-Lactate Concentration in Cell Culture Supernatants)

  1. Prepare 5.0×104 HeLa cells and treat overnight with 100 µM Phloretin (Product No. P1966) and 25 µM Rotenone (Product No. R0090).
  2. Collect the supernatant.
  3. Dilute the Lactate Standard Solution (10 mM) supplied in the kit with ultrapure water to prepare each dilution series. (1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, 0.015625 mM, 0 mM)
  4. Add 20 µL of Lactate Standard Solutions and sample solutions to separate wells of a 96 well plate.
  5. Prepare the working solution, add 80 µL of Working Solution to each well.
  6. Incubate the microplate at 37 °C for 30 minutes.
  7. Measure the absorbance at 450 nm by using a microplate reader.
  8. Generate a calibration curve using the data from the L-lactate dilution series after subtracting the blank. (Figure 2)
  9. Determine the concentration of L-lactate in the sample using a calibration curve. (Figure 3)
Calibration curve of L-lactate concentration
Figure 2. Calibration curve of L-lactate concentration
Concentration of L-lactate in cell culture supernatants after treatment with Phloretin and Rotenone
Figure 3. Concentration of L-lactate in cell culture supernatants after treatment with Phloretin (Product No. P1966) and Rotenone (Product No. R0090)
Phloretin reduces glucose uptake, leading to decreased lactate levels in the culture supernatant.
Rotenone inhibits mitochondrial complex I, resulting in increased lactate levels due to enhanced glycolysis.

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Example of use 2 (Quantification of L-Lactate Concentration in Cells)

  1. Collect HeLa cells (1.0×105 cells) treated overnight with 50 µM α-CHCA (Product No. C1768).
  2. Centrifuge at 300×g for 2 minutes and remove the supernatant.
  3. Add 300 µL of cold PBS, suspend by pipetting, centrifuge at 300 × g for 2 minutes, and remove the supernatant.
  4. Add 300 µL of cell lysis solution (0.1% Triton-X) and vortex for 1 minute to create a cell lysis solution.
  5. Centrifuge at 8000 × g for 5 minutes and collect the supernatant.
  6. Transfer 200 μL of the solution from step 5 to an ultrafiltration membrane filter (molecular weight cut-off: 10K), centrifuge at 12,000×g for 10 minutes, collect the filtrate, and use it as the measurement sample.
  7. Dilute the Lactate Standard Solution (10 mM) supplied in the kit with ultrapure water to prepare each dilution series. (1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, 0.015625 mM, 0 mM)
  8. Add 20 µL of Lactate Standard Solutions and sample solutions to separate wells of a 96 well plate.
  9. Prepare the working solution, add 80 µL of Working Solution to each well.
  10. Incubate the microplate at 37 °C for 30 minutes.
  11. Measure the absorbance at 450 nm by using a microplate reader.
  12. Generate a calibration curve using the data from the L-lactate dilution series after subtracting the blank. (Figure 2)
  13. Determine the concentration of lactate in the sample using a calibration curve. (Figure 4)
Intracellular L-Lactate concentration after treatment with α-CHCA
Figure 4. Intracellular L-Lactate concentration after treatment with α-CHCA (Product No. C1768)
α-CHCA inhibits lactate export, resulting in increased intracellular lactate levels.

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Product Brochure

L-Lactate Assay Kit (PDF file)

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Related Products

G0656
Glucose Measurement Kit
N1178
2-NBDG
P1966
Phloretin
R0090
Rotenone
C1768
α-CHCA
D0051
2-Deoxy-D-glucose

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Mitochondrial Membrane Potential Measurement Reagent

JC-1 Mitochondrial Membrane Potential Assay Kit (Product No. J0043) has been designed to measure mitochondrial membrane potential through the action of JC-1, a fluorescent dye that accumulates in mitochondria in a membrane potential-dependent manner. In healthy mitochondria with an intact membrane potential, JC-1 forms aggregates and emits red fluorescence, whereas in mitochondria with a decreased membrane potential, JC-1 exists as a monomer and emits green fluorescence. In this way, changes in the intensity ratio of red and green fluorescence can be used to evaluate the functional status of mitochondria.

Product

J0043
JC-1 Mitochondrial Membrane Potential Assay Kit

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Kit Components

  • JC-1 Dye 100 nmol×1 vial
  • CCCP Solution (10mM in DMSO) 500 μL×1 vial

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Example of use 1 (Fluorescence Microplate Reader)

  1. Seed cells into a dish or a chamber slide and incubate at 37 °C in a 5% CO2 incubator (4.0×104 cell/mL, 200 μL/well). Incubate for two nights.
  2. Remove the medium and add 200 µL/well of 50 µM and 100 µM CCCP (Product No. C2876) solutions diluted in medium. Incubate for 1.5 hours in a 5% CO2 incubator.
  3. Remove half of the medium from each well (both samples and CCCP controls). Add to each well an equivalent amount of 10 µM JC-1 working solution. Incubate the cells at 37 °C in a 5% CO2 incubator for 20 minutes.
  4. Remove medium from wells, and wash 2x with PBS.
  5. Add 200 µL of PBS and measure fluorescence intensity via a plate reader.
    Green：Ex 475 - 495 nm / Em 528 - 552 nm
    Red：Ex 528 - 552 nm / Em 585 - 595 nm
Mitochondrial membrane potential in cells treated with CCCP, measured using a plate reader
Figure 5. Mitochondrial membrane potential in cells treated with CCCP (Product No. C2876), measured using a plate reader.
CCCP induced a concentration-dependent reduction in mitochondrial membrane potential.

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Example of use 2 (Fluorescence Microscopy)

  1. Seed cells into 8wellchamber slide and incubate at 37 °C in 5% CO2 incubator (2.5×105 cell/mL, 200 μL/well). Incubate for over nights.
  2. Remove the medium and add 200 µL/well of 50 µM and 100 µM CCCP (Product No. C2876) solutions diluted in medium. Incubate for 1.5 hours in a 5% CO2 incubator.
  3. Remove half of the medium from each well (both samples and CCCP controls). Add to each well an equivalent amount of 10 µM JC-1 working solution. Incubate the cells at 37 °C in a 5% CO2 incubator for 20 minutes.
  4. Remove medium from wells, and wash 2x with PBS.
  5. Add 200 µL of PBS and observe the cells under a fluorescence microscope.
    Green： Ex 450 - 490nm / Em 500 - 550 nm
    Red： Ex 538 - 562 nm / Em 570 - 640 nm
Mitochondrial membrane potential in cells treated with CCCP, observed by fluorescence microscopy
Figure 6. Mitochondrial membrane potential in cells treated with CCCP (Product No. C2876), observed by fluorescence microscopy.
CCCP induced a concentration-dependent reduction in mitochondrial membrane potential.

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Product Brochure

Mitochondrial Membrane Potential Assay Kit (PDF file)

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Other Mitochondrial Membrane Potential Detection Reagents

T4300
JC-1
T3608
Tetramethylrhodamine Methyl Ester Perchlorate

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Related Products

I1265
Intracellular Reactive Oxygen Species (ROS) Detection Assay Kit
C2876
CCCP
C3463
FCCP
M2009
Metformin Hydrochloride
R0090
Rotenone

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Related Product Category Page

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References

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