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.

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＞

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.

If the microplate is incubated with an incubator (or heat block, thermostatic chamber, etc.) after adding reagents and Mineral Oil, the temperature difference in the plate reader will affect the OCR result. This leads to a decrease in data reproducibility. Therefore, please use a temperature-controllable plate reader.

＜General Protocol＞

Step 3 and 7 for floating cells; Step 5 and 9 for adherent cells in the manual.

＜Effect of incubation environment on results＞

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

(Reference)

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.

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

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

Please check the following two experimental conditions.

(1) If the temperature varies during measurement, it may affect the OCR result. Please make sure the following two steps are performed exactly according to the manual. 
　 ・The Mineral Oil, medium, and medium-diluted sample solutions should be preheated to around  37°C before use.
　 ・After adding the reagent and Mineral oil, please use a temperature-controllable plate reader to incubate.
　　 Please refer to the FAQ "Why temperature-controllable plate readers are highly recommended for this kit?".

(2) Optimizing the cell number before the final examination is recommended. If the cell number is low, the difference between the experimental and control groups may not be significant.

  　 【The OCR value with cell numbers and reagent treatment (Concept image)】