Glycolysis/OXPHOS Assay Kit

Glycolysis/OXPHOS Assay Kit

Glycolysis/Oxidative phosphorylation Assay

  • Easy test via plate reader, no need for expensive equipment
  • All reagent acquired is included, ready to use kit
  • Easy-to-understand detailed protocol
  • Product code
    G270  Glycolysis/OXPHOS Assay Kit
Unit size --
50 tests Please inquire distributors about price.


The approximate usage depends on the experiment.
Please refer to the Q&A How many samples can be tested by this kit?.

Component
50 tests ・Dye Mixture
・Lactate Standard
・LDH Solution
・Lactate Assay Buffer
・Reconstitution Buffer
・Substrate
・Luciferase Solution
・ATP Assay Buffer
・Oligomycin
・2-DG
×1
150 µl×1
12 µl×1
5.5 ml×1
550 µl×1
×1
10 µl×1
5.5 ml×1
×1
140 µl×1

Description

   Many cancer cells produce ATP through energy metabolism that depends on the glycolytic pathway. On the other hand, it has been recently reported that cancer cells whose glycolytic pathway is suppressed survive by shifting their energy metabolism to oxidative phosphorylation(OXPHOS). These phenomena has attracted much attention because it will not only help elucidate the mechanisms of anticancer drugs but also lead to the development of therapeutic strategies in various diseases including aging and neurodegenerative disorders.
 This kit evaluates glycolytic capacity, metabolism shift, and whether cells rely on the glycolytic system or oxidative phosphorylation for energy production using a plate reader. This ready-to-use kit includes all the reagents acquired.

Manual

Technical info

  This kit measures the concentration of intracellular adenosine triphosphate (ATP) by the luciferase luminescence method and the amount of lactate released from cells by the absorbance method using water soluble tetrazolium (WST) formazan. Using the inhibitors contained in this kit, it is possible to evaluate a shift in the intracellular metabolism between glycolysis and oxidative phosphorylation (OXPHOS). In addition, this kit can be used with microplates.

Three Applications

  The inhibition of ATP synthesis via OXPHOS by oligomycin or inhibition of ATP synthesis via glycolysis by 2-deoxy-D-glucose (2-DG) can be determined by measuring changes in the amounts of ATP (luminescence value) and lactate (absorbance value), respectively, which can be used to evaluate the following three pathways.

Evaluation 1: Glycolytic capacity

  Oligomycin is used to inhibit ATP synthesis by OXPHOS, and then the potential of the glycolytic pathway using lactate production as an indicator can be evaluated.

<Application Data>
  The glycolytic capacity of HeLa cells treated with a glycolytic inhibitor (2-DG concentration: 2 mmol/l) was compared with that of untreated HeLa cells (control). When ATP synthesis by OXPHOS was inhibited by oligomycin, the glycolytic pathway was enhanced in the control cells, but not in cells in which the glycolytic pathway was inhibited.

*For more details, please refer to General Protocol ① (Analysis of Cellular Glycolytic Capacity) on page 3 of the manual.

Evaluation 2: Metabolism shift

  Measuring the ATP levels in oligomycin-treated cells and control cells can be used to calculate the amount of ATP synthesized by mitochondria, which is an indicator of a shift in cellular metabolism.

<Application Data>

  We evaluated whether treatment with a glycolytic inhibitor (2-DG concentration: 2 mmol/l) would cause a metabolic shift in HeLa cells. The ATP production capacity of the control cell group was dependent on the glycolytic pathway, whereas inhibition of the glycolytic pathway in HeLa cells shifted ATP production from glycolysis to OXPHOS and increased ATP production in the mitochondria.

*For more details, please refer to General Protocol ② (Analysis of Metabolism Shift) on page 5 of the manual.

Evaluation 3: Metabolic pathway dependence

  Combining methods (1) and (2) can be used to measure the metabolic pathway dependency of cells.
  Cells are treated with oligomycin or 2-DG to inhibit OXPHOS or ATP synthesis in the glycolytic pathway, and the amounts of ATP and lactate production are measured, respectively. Changes in the amount of ATP can be used to determine the efficiency of energy production, and changes in the amount of lactate produced can be used to determine changes in glycolytic capacity and evaluate whether cells are dependent on glycolysis or OXPHOS.

<Application Data>
 We evaluated the metabolic pathway dependence of HeLa cells treated with a glycolytic inhibitor (2-DG concentration: 2 mmol/l).
  ATP levels in the cells without glycolytic system inhibition treatment were markedly reduced by 2-DG treatment but were unchanged by oligomycin treatment. The amount of lactate production was increased by oligomycin treatment. This indicates that metabolism in HeLa cells in the absence of glycolytic inhibitor treatment is highly dependent on the glycolytic system.
  In contrast, oligomycin treatment did not increase lactate production in the glycolytic inhibitor-treated cells, and ATP levels were markedly reduced by oligomycin treatment. In addition, the amount of ATP was decreased significantly after oligomycin treatment, indicating that the metabolism of glycolysis-inhibited HeLa cells depended on OXPHOS.

*For more details, please refer to General Protocol ③ (Analysis of Metabolic Pathway Dependence) on page 8 of the manual.

Q & A

Q

How many samples can be tested by this kit?

A

Analyzing samples and standards in triplicate is recommended for accuracy. The number of samples that can be measured is shown in the table below, assuming that each independent sample is measured in triplicate.

  50 tests
Lactate Assay ATP Assay
Glycolysis Metabolism Shift Metabolic pathway
Dependence
Glycolysis Metabolism Shift Metabolic pathway
Dependence
96 well plate 48 well   48 well   48 well 48 well
Number of Samples
(n=3)
6 samples   4 samples   8 samples 5 samples


*This is the maximum number of samples that can be measured without pre-experiment.
*If the medium contains serum, it is recommended to measure the lactate concentration in the medium as a background control for the lactate assay.

  50 tests
Lactate Assay ATP Assay
Glycolysis Metabolism Shift Metabolic pathway
Dependence
Glycolysis Metabolism Shift Metabolic pathway
Dependence
96 well plate 9 well   9 well   21 well 9 well
Number of Samples
(n=3)
5 samples   4 samples   4 samples 4 samples

*This is the maximum number of samples that can be measured with pre-experiment.
*If the medium contains serum, it is recommended to measure the lactate concentration in the medium as a background control for the lactate assay.

 


Glycolytic Capacity Evaluation (Lactate Assay); Example of plate layout (n=3)
(Left: Without pre-experiment; Right: With pre-experiment)

Metabolism Shift Evaluation (ATP Assay); Example of plate layout (n=3)
(Left: Without pre-experiment; Right: With pre-experiment)

Metabolic Pathway Dependence Evaluation; Example of plate layout (n=3)
(Left: ATP Assay; Right: Lactate Assay)(Without pre-experiment)

Metabolic Pathway Dependence Evaluation; Example of plate layout (n=3)
(Left: ATP Assay; Right: Lactate Assay)(With pre-experiment)

Q

In the glycolysis assay, the absorbance of samples did not differ from that of the blank, what is the possible reason?

A

The amount of lactate released from the cells might be low. Try increasing the number of cells or extending the incubation time from 3 hours to 5 hours.

Q

Do I need to normalize the lactate and ATP concentrations, by using a protein quantification assay?

A

Because the number of cells before and after drug treatment can change depending on the cell type or drug, it is recommended to confirm the change via a protein quantification method, in advance. Protein quantification cannot be performed in the same wells as the ATP assay or lactate assay. Prepare separate wells for protein quantification. 

For reference, in the case of HeLa cells treated with oligomycin and 2-DG for 5 hours, there was little difference in the number of cells before and after treatments as assessed by the protein quantification method.

Q

What other filter can be used besides a 450 nm filter?

A

You can use filters with an absorbance between 450 nm and 490 nm. However, for the longer wavelengths, the absorbance will be lower than that measured at 450 nm.

Q

Can I measure samples or drugs that contain a reducing agent?

A

No, you will not be able to accurately measure the lactate concentration, because WST reacts with reducing agents and increases the background. However, if the samples must contain a reducing agent, the reagent blank should be measured as a background control.

Q

Can the working solution be stored?

A

The working solution cannot be stored for long periods; it should be prepared prior to use. The working solution is stable for 4 hours at room temperature when protected from light.

Q

How long is the luminescence signal stable?

A

The luminescence signal is stable for 3 hours. If you are not able to measure immediately after reagent addition, store the microplate at 25 ℃ protected from light until measurement.

Q

What other microplates can be used besides a white microplate?

A

We recommend a white microplate. The luminescence signal with a black microplate tends to be lower than with a white microplate.

Handling and storage condition

Handling and storage condition
1. Store at 0-4℃; 2. Protect from light
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