We recommend 490 nm, but a difference of 490±2-3 nm is no problem. 450 nm filters can be used as we have experience with them. Please note that the absorbance will be higher than with the 490 nm filter.

Cytotoxicity LDH Assay Kit-WST and Cell Counting Kit-8 do not necessarily correlate with each other due to their different measurement principles. Therefore, when testing for cytotoxicity, it is recommended to use several types of indicators, such as the LDH Assay and the Cell Counting Kit-8.
 - Cytotoxicity LDH Assay Kit-WST: Measures LDH activity released into the medium due to cell membrane damage (cell membrane damage is an indicator).
 - Cell Counting Kit-8: Measures intracellular metabolic activity (dehydrogenase activity is an indicator).

The LD50 value may be affected by the test substance depending on the state of the cells. The LD50 value is affected by the state of the cells.
The LD50 value may be more reliable if a narrower dilution factor is used when preparing a dilution series of the test substance.
It is recommended that the concentration of the test substance to be used be determined according to the following procedure.

＜Study procedure＞

(1) Repeat the 10-fold dilution based on the highest available concentration of the test substance and perform toxicity testing over a wide sample concentration range of about 5-6 digits. [Figure 1]. Using the operation in (1), make a rough estimate of the highest predicted concentration in the concentration range at which toxicity will occur.
(2) Based on the result of (1), repeat 2-fold dilution based on the predicted maximum concentration (the lowest concentration that causes 100% lethality) at which the concentration of the substance to be tested appears, and perform toxicity testing in a concentration range covering 2-3 orders of magnitude. [Figure 2].
(3) Repeat step (2) to finally establish a concentration range in which at least 3 sample concentrations between 20-80% cytotoxicity can be plotted. [Figure 3] 

This product is designed to measure cytotoxicity using total LDH activity as an indicator, so it cannot measure LDH1, LDH2, LDH3, etc. separately. 

It can be stored for approximately one week in refrigerated storage¹⁾.
Storage at room temperature²⁾ or freezing is not recommended due to inactivation of LDH.

¹⁾ M. Allen, P. Millett, E. Dawes, N. Rushton., "Lactate dehydrogenase activity as a rapid and sensitive test for the quantification of cell numbers in vitro.", Clin Mater. 1994; 16, (4), 189.
²⁾ A. Wagner, A. Marc, JM. Engasser, A. Einsele, "The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of Biotechnol Bioeng. 1992; 39, (3), :320.

Stability may vary depending on evaluation conditions and other factors. Please refer to the above paper with understanding.

No, LDH standards are not included in the kit as the kit is intended for cytotoxicity testing.

Yes, the assay should be performed within 3 hours after the addition of the Stop Solution. In this case, please protect the plate from light.

There is a lot of variation between wells, what could be the cause?

Please check the following possible reasons.

(1) Air bubbles are formed on the surface of the culture medium.

Please remove the air bubbles using the following methods.
Remove air bubbles with the tip of a syringe or syringe needle.
(If you have a 96-well plate centrifuge) Centrifuge at 1,000 x g for 1 minute.

(2) The reagent concentration has changed due to evaporation of water from the culture medium during incubation.

The outermost wells of the microplate are prone to water evaporation during incubation. When incubating for a long time, do not use the outermost wells for measurement, but use only the culture medium in them.

(3) When adding reagents, there is a large time difference between the first and last wells to be added.

For Working Solution and Stop Solution, which are added to all wells, the use of a multichannel pipette is recommended.

(4)The added reagents are not well mixed.

When adding Lysis Buffer, Working Solution and Stop Solution, mix them with a plate mixer or gently tap the side of the plate with your finger to mix the added reagents and medium.
Note that the amount of Lysis Buffer added is small and will affect the High Control value.
When tapping the plate, be careful not to spill the medium in the wells.

(5) The weight of the (multichannel) pipette used is not accurate.

Please check if the pipette weight is accurate.

The following causes are possible.
(1) LDH present in serum in the medium is increasing the background. Use serum-free medium or medium prepared with 5% or less serum.
(2) Strong reducing substances in the test substance or medium may cause the WST dye to miscolorize and increase the background. Confirm this by measuring a reagent blank.
(In addition, common culture media such as DMEM, RPMI, F-12, etc. usually do not contain reducing agents).

(1) The absorbance of the low control may be higher due to high background. Check the following

LDH in serum in the medium increases the background. Use serum-free medium or medium prepared with 5% or less serum.
If the test sample or culture medium contains strong reducing substances, the WST dye will mischromatize and increase the background. Confirm this by measuring a reagent blank.

(2) The High Control may contain viable cells.

To ensure that the Lysis Buffer is evenly distributed in the wells and that all cells are dead cells, gently vortex the plate after adding the Lysis Buffer to mix the Lysis Buffer thoroughly.