01 Cell Proliferation / Cell Cytotoxicity Assay

Extracellular OCR Plate Assay Kit

Extracellular OCR Plate Assay Kit

Oxygen Consumption Rate(OCR) Plate Assay Kit

  • Applicable to regular fluorescent plate reader with temperature-controlled incubation
  • No need for an expensive instrument, special medium, and plates
  • All-in-One Kit with OCR calculation Sheets
  • Product code
    E297  Extracellular OCR Plate Assay Kit
Unit size Price Item Code
100 tests $380.00 E297-10

Note: A plate reader with temperature-controlled incubation is required to measure oxygen consumption.

Please check the "Precaution" before purchase and use.

Order
100 tests
Quantity may be adjusted within the cart.
Component
100 tests ・Oxygen Probe
・Mineral Oil
110 µl×1
10 ml×1

OCR is an Important Indicator for Mitochondrial Function

 Since oxygen is consumed mainly in the process of adenosine triphosphate (ATP) production by mitochondrial oxidative phosphorylation, its oxygen consumption rate (OCR) is an indicator for the analysis of mitochondrial function. It is known that cancer cells produce ATP using the glycolytic pathway, which is less efficient than oxidative phosphorylation. It is also known that in immune cells, the predominance of oxidative phosphorylation inhibits anti-tumor effects, whereas the predominance of the glycolytic pathway promotes anti-tumor effects. As an indicator of their energy metabolism, the OCR of cells has been measured.

Technical info

The Extracellular OCR Plate Assay Kit includes an Oxygen Probe, which has the property of increasing phosphorescence intensity as the oxygen concentration in the medium decreases, and Mineral Oil blocks the influx of oxygen from the air.
After measuring the phosphorescence intensity according to the extracellular oxygen concentration with a fluorescent microplate reader, the OCR of the cells is calculated (automatic calculation sheet) based on the Stern-Volmer equation.

*This product was commercialized under the guidance of Dr. Toshitada Yoshihara of Gunma University.

Comparison with Existing Method

Until now, OCR measurement required expensive devices such as flux analyzers and plate readers with time-resolved functions, as well as optional functions for plate readers, periments. This kit is recommended for those who are just starting experiments, as it can be used with a regular fluorescent microplate reader and comes a complete package of all necessary reagents.

  Dojindo Laboratories
Extracellular OCR Plate Assay Kit
Company A's Product Company B's Instrument
Instrument Fluorescence
Microplate Reader
(Temperature control function is needed)
Time-Resolved Fluorescence
Microplate Reader
(Temperature control function is needed)
Flux Analyzer
Principle Phosphorescence Phosphorescence Phosphorescence
Wavelength Ex=500 nm / Em=650 nm  Ex=380 nm / Em=650 nm  Ex=532 nm / Em=650 nm
Required Special
Reagents
No No Special Plates,
Special Medium
Added Reagents 1 type/ well  1 type/ well Up to 4 types/ well
(Continuous adding)
Assay Category OCR OCR OCR & hydrogen
ion concentrations

Comparison with Flux Analyzer

Flux Analyzer (XFe24) and this kit were measured on the same day under the same conditions (cell type, cell number, and FCCP concentration).
As a result, correlated data of oxygen consumption rate changes were obtained for XFe24 and this kit.


 
 Cells: HepG2
 Cell Number: 5×104 cells/well
 Stimulation: FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)
 FCCP Concentration: 2 μmol/l

Application Data: Evaluation of Maximum Respiratory Capacity of Cells

In HepG2 cells, the maximal respiration of cells was evaluated from the change in OCR values upon FCCP stimulation.

OCR was measured at 2 µmol/l and 4 µmol/l of FCCP. A decrease in OCR was observed at 4 µmol/l compared to 2 µmol/l, indicating maximal respiration at 2 µmol/l of FCCP.

 
 Cells: HepG2
 Cell Number: 5×104 cells/well
 Stimulation: FCCP
 FCCP Concentration: 2, 4 μmol/l

Application Data: Inhibition of Mitochondrial Electron Transport Chain

Antimycin stimulation of Jurkat cells was used to evaluate the changes in cellular state upon inhibition of the mitochondrial electron transport chain using a variety of indicators.

The results showed that inhibition of the electron transport chain resulted in (1) a decrease in mitochondrial membrane potential and (2) a decrease in OCR. In addition, (3) the NAD+/NADH ratio of the entire glycolytic pathway decreased due to increased metabolism of pyruvate to lactate to maintain the glycolytic pathway, (4) GSH depletion due to increased reactive oxygen species (ROS), and (6) increase in the NADP+/NADPH ratio due to decreased NADH required for glutathione biosynthesis were observed. 

Q & A

Q

How many samples can be tested by this kit?

A

24 samples can be measured when the same number of cells of one cell type is tested.

*If more than two cell types or multiple cell numbers are used in an experiment, separate Blanks and Controls must be prepared, and the number of samples that can be measured will vary. For details, please refer to the examples of plate layouts according to the  manual.

Q

Is there any applications for floating cells?

A

We prepared an example of Jurkat cells experiment.
<Protocol>
(1) Jurkat cells (3.0×106 cells/ml) were suspended in RPMI medium as Blank 3 and Jurkat cells (3.0×106 cells/ml) were suspended in working solution as Control or Sample. The cells were seeded in 100 µl (300,000 cells/well) in 96-well black clear-bottom microplates.
(2) 100 µl of RPMI medium was added to Blank 1 and 100 µl of working solution to Blank 2.
(3) Microplates were placed in a plate reader pre-set at 37°C and incubated for 30 minutes.
(4) Add 10 µl of RPMI medium to each of Blank 1, Blank 2, Blank 3, and Control.
(5)Sample solution (Antimycin or FCCP solution) diluted with RPMI medium was added to Sample in 10 µl portions.
(6) One drop of Mineral Oil was added to each well immediately after the addition of the Sample solution.
(7) The microplate was placed in a plate reader set at 37°C and incubated for 5 minutes.
(8)Intensities were measured with a fluorescent plate reader every 10 min for 200 min on a time course (Ex: 500 nm, Em: 650 nm, Bottom reading).
(9) OCR values were calculated by entering the intensity values obtained into a downloaded dedicated Excel calculation sheet. 

Amounts of sample and reagent required for each well.

<Result>


         
 

Q

Please tell me how to calculate OCR with this kit.

A

Please use the Excel Calculation Sheet and follow the instructions below.

<Outline of OCR calculation procedure>

(1) Input the intensity values obtained from the OCR measurement into the calculation sheet, and the oxygen content (nmol) is automatically calculated using the Stern-Volmer formula.
(2) From the graph of time (min) vs. oxygen content (nmol), check the range of linearity obtained for all conditions measured.
(3) Calculate the slope over the range of time (min) and oxygen content (nmol) confirmed in step(2).
(4) Calculate OCR (pmol/min) from the slope calculated in step (3).
For details, please refer to "Analysis" in the manual.

Q

Does Mineral Oil have cytotoxicity to cells?

A

No toxicity was observed in cells treated with Mineral oil when measured by Cell Counting Kit-8 cytotoxicity assay.

(Reference)


 

Q

How to measure the cell number after OCR assay

A

Using nucleic acid probe (Code: H342)Hoechst 33342 to measure the cell number per well, here is an example of the protocol.

<Protocol>
(1) Seed cells into the wells for OCR measurement (liquid volume: 100 μl/well).
(2) Cells prepared for calibration curve preparation were seeded into the wells (liquid volume: 100 μl/well).
(3) OCR was measured according to the instruction manual.
(4) Add 10 µl/well of medium to the wells for calibration (to align the volume of medium to 110 µl/well with that of the wells for OCR measurement).
(5) Hoechst 33342 solution (10 µg/ml) diluted with the medium was added to all wells at 100 µl/well.
  *Wells for OCR measurement was added from the top of the oil.
(6) Incubated at 37°C for 30 minutes.
(7) Measured with a fluorescence plate reader (Ex: 350 nm, Em: 461 nm ).
(8) A calibration curve (X-axis: number of cells, Y-axis: fluorescence intensity) was prepared and the number of cells in the wells for OCR measurement was calculated.

Q

Can I store the working solution for a long period of time?

A

The Working solution cannot be stored. Prepare it as needed.

Q

Will repeated freezing and thawing of Oxygen Probe or Mineral Oil affect the assay?

A

We have confirmed that repeated freeze-thaw cycles of  Oxygen Probe and Mineral Oil have no effect on the assay.

Handling and storage condition

Handling and storage condition
Store at -20 °C
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