The number of samples that can be measured is shown in the table below, assuming that each independent sample is measured in triplicate.
(Lactate Assay)

*If the lactate concentration of the sample is unknown, perform a preliminary experiment to determine the dilution factor so that the absorbance is less than that of the 1 mmol/L lactate standard solution. Refer to "Sample preparation" in the instruction manual.
*The maximum number of samples that can be measured without a preliminary experiment or with a preliminary experiment.
*When performing the Lactate Assay, if the medium contains serum, it is recommended that a test sample be prepared only from the medium containing the serum used as the background control.

Plate Layout for the Lactate Assay (n=3)
(Left: without preliminary experiment, Right: with preliminary experiment)

(JC-1 Assay)

This kit contains JC-1 Assay reagents for 48 wells of 96-well microplates.

The experimental procedure and approximate time required for each assay is shown in the figure below.

If the lactate assay is performed first, the difference in measurement time may affect the results depending on the stimulation conditions. Be sure to perform the JC-1 assay first after collecting the supernatant. Cell culture supernatants can be stored frozen (-20°C) for up to 1 month.

You can use filters with an absorbance between 450 nm and 490 nm. However, the absorbance value can become lower if the sample is not measured at 450 nm.

You can’t measure an accurate Lactate level because WST dye interferes with the reducing agent. If you add test material which possesses reducing properties, please prepare a few wells that only contain the medium without cells and only test material for background control. Please subtract the background from the absorbance of standard curve or samples.

You can’t store the Working solution. Please prepare the Working solution prior to use. Please protect from light because the Working solution is light-sensitive. The Working solution is stable for 4 hours at room temperature with protection from light.
Once the Working solution is exposed to light, the color of the solution becomes from red to orange. It caused to increase background.

Can be stored frozen (-20°C) for one month.

Serum-containing medium can be used for cell washing and preparing JC-1 working solution. For fluorescence observation, we recommend the use of Imaging Buffer solution, but if serum-containing medium is used, phenol red-free medium is recommended.

Fixation is not recommended because the JC-1 dye does not work well due to mitochondrial depolarization.

The cause may be that the amount of lactic acid released from the cells is low. Please increase the number of cells to be seeded or extend the incubation time further.

Calculate the ratio of red fluorescence value/green fluorescence value for each of the drug-treated samples and the control. The lower the fluorescence ratio, the lower the mitochondrial membrane potential.

(Reason for evaluating by red/green ratio)

　Since JC-1 accumulates in cells in a membrane potential-dependent manner, the concentration of JC-1 per cell may differ depending on the cell condition^{1), 2)} (the accumulation concentration of JC-1 is different between control and drug-treated samples due to the different cell conditions).

In addition, JC-1 aggregates and shifts its fluorescence from green to red when the mitochondrial membrane potential is high. The amount of this aggregation depends on the degree of membrane potential³⁾, allowing comparison of mitochondrial membrane potential between samples in terms of the red/green ratio.

[Reference]
1) A. Cossarizza, et al., Biochem Biophys Res Commun. 1993, 197(1), 40.

2) A. Perelman, et al., Cell Death and Disease, 2012, 3, e430.

3) S. T. Smiley, et al, Proc. Nail. Acad. Sci. 1991, 88, 3671.

Glycolysis/JC-1 MitoMP Assay Kit is a combined product of Lactate Assay Kit-WST (product code: L256) and JC-1-MitoMP Detection Kit (Product Code: MT09).
Please order these single products in combination for the larger unit size.

Please note that the components themselves of these products are the same, although some component names in the L256 appear slightly different from this combined product.