Cytotoxicity LDH Assay Kit-WST

Cytotoxicity LDH Assay Kit-WST

Cytotoxicity Assay

  • Can be used with and without transferring supernatant
  • Stable Working Solution (6 months at 0-5 oc)
  • No need to freeze the Working Solution
  • Product code
    CK12  Cytotoxicity LDH Assay Kit-WST
Unit size --
100 tests Please inquire distributors about price.
500 tests Please inquire distributors about price.
2000 tests Please inquire distributors about price.
Component
100 tests ・Dye Mixture
・Assay Buffer
・Lysis Buffer
・Stop Solution
×1
11 ml×1
1.1 ml×1
5.5 ml×1
500 tests ・Dye Mixture
・Assay Buffer
・Lysis Buffer
・Stop Solution
×1
55 ml×1
5.5 ml×1
27.5 ml×1
2000 tests ・Dye Mixture
・Assay Buffer
・Lysis Buffer
・Stop Solution
×4
55 ml×4
5.5 ml×4
27.5 ml×4

Detection Principle

Lactate dehydrogenase(LDH) is an enzyme that presents in almost cell types and it catalyzes the oxidation of lactate to pyruvate in the presence of co-enzyme NAD+. Once cells are impaired by stress, injuries, chemicals, or intercellular signals, LDH is rapidly released from the cell membrane. Thus, the measurement of the amount of released LDH from cells is one of the major methods to assess the cell death. Since Dojindo’s Cytotoxicity LDH Assay Kit-WST neither reflects the activity of living cells nor is harmful to cells, it allows the assay to perform in wells containing both viable and damaged cells.

Choose between Two Procedures

Cytotoxicity LDH Assay Kit-WST can be applied with and without supernatant transferring. Please choose suitable method for your experiment.

Stable Working Solution

Working Solution is stable for 6 months under refrigerated conditions. Therefore, after the preparation, Working Solution can be used as a ready-to-use solution at any time during this period.

Experimental Examples

To obtain an accurate result in cytotoxicity assay, the samples are measured by different principles. Cell Counting Kit-8 (Product Code: CK04) measure NADH in living cells and LDH assay measure released LDH from dead cells. According to these results, the living cells decreased and dead cells increased with higher concentration of toxicant.

Developer Dojindo Molecular Technologies, Inc.

Technical info

Reconstituted Working Solution has higher stability than competitor’s kit, eliminating the additional time to prepare the Working Solution for each assay.

Comparison of Working Solution Stability

Linearity

Dojindo’s Cytotoxicity LDH Assay Kit-WST has higher sensitivity than competitor’s kits

Cytotoxicity of mitomycin C using HeLa cells

Culture medium: MEM, 10% FBS
Incubation: 37C, 5% CO2, 48 hours

References

Open References

Reference No. Sample Citation
1) Cell
(C3H10T1/2)
S. F. Jin, H. L. Ma, Z. L. Liu, S. T. Fu, C. P Zhang, Y. He, "XL413, a cell division cycle 7 kinase inhibitor enhanced the anti-fibrotic effect of pirfenidone on TGF-β1-stimulated C3H10T1/2 cells via Smad2/4."Exp Cell Res., 2015, 339, (2), 289.
2) Cell
(HepG2)
S. Watanabe, C. S. Moniaga, S. Nielsen, M. Hara-Chikuma, "Aquaporin-9 facilitates membrane transport of hydrogen peroxide in mammalian cells."Biochem Biophys Res Commun ., 2016, 471, 191.
3) Cell
(HEECs, Human Esophageal Epithelial Cells)
L. Wu, T. Oshima, J. Shan, H. Sei, T. Tomita, Y. Ohda, H. Fukui, J. Watari, H. Miwa , "PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells."Am J Physiol Gastrointest Liver Physiol ., 2015, 309, G695.
4) Cell
(Human L02 Hepatocyte)
D. Zhou, B-H Li, J. Wang, Y-N Ding, Y. Dong, Y-W Chen and J-G Fan, "Prolyl Oligopeptidase Inhibition Attenuates Steatosis in the L02 Human Liver Cell Line."PloS ONE., 2016, 11, (10), e0165224.
5) Cell
(Human primary Hepatocyte)
T. Fukami, A. Iida, K. Konishi, M. Nakajima , "Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity"Biochem. Pharmacol., 2016, 116, 153.
6) Cell
(Mouse embryonic fibroblast:MEF)
Y. Takanezawa, R. Nakamura, Y. Sone, S. Uraguchi and M. Kiyono, "Atg5-dependent autophagy plays a protective role against methylmercury-induced cytotoxicity"Toxicol. Lett., 2016, 262, 135.
7) Cell
(neural stem/progenitor cells)
M. Tanaka, M. Yoneyama, T. Shiba, T. Yamaguchi, K. Ogita, "Protease-activated receptor-1 negatively regulates proliferation of neural stem/progenitor cells derived from the hippocampal dentate gyrus of the adult mouse "J Pharmacol Sci., 2016, 131, (3), 162
8) Cell
(Primary cultured cortical neurons)
S. Wakatsuki, A. Furuno, M. Ohshima, and T. Araki, "Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration"J Cell Biol., 2015, 211, (4), 881.
9) Cell
(DLD1 and AGS cells)
A. Ishijima, K. Minamihata, S. Yamaguchi, S. Yamahira, R. Ichikawa, E. Kobayashi, M. Iijima, Y. Shibasaki, T. Azuma, T. Nagamune & I. Sakuma, "Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro"Scientific Reports ., 2017, DOI:10.1038/srep44077.
10) Cell
(U87, HUVEC cell)
N. Li, W. Zhang, M. Khan, L. Lin, JM. Lin, "MoS2-LA-PEI nanocomposite carrier for real-time imaging of ATP metabolism in glioma stem cells co-cultured with endothelial cells on a microfluidic system"Biosens Bioelectron., 2017, 99, (2018), 142.
11) Tissue
(Mouse liver)
Y. Sakurai, W. Mizumura, M. Murata, T. Hada, S. Yamamoto, K. Ito, K. Iwasaki, T. Katoh, Y. Goto, A. Takagi, M. Kohara, H. Suga, and H. Harashima, "Efficient siRNA delivery by lipid nanoparticles modified with a non-standard macrocyclic peptide for EpCAM-targeting"Mol. Pharm.., 2017,10.1021/acs.molpharmaceut.7b00362.
12) Cell
(Human pleural mesothelial cell)
K. Hattori, K. Nakadate, A. Morii, T. Noguchi, Y. Ogasawara, K. Ishii., "Exposure to nano-size titanium dioxide causes oxidative damages in human mesothelial cells: The crystal form rather than size of particle contributes to cytotoxicity."Biochem. Biophys. Res. Commun.., 2017,10.1016/j.bbrc.2017.08.054.
13) Tissue
(murine distal colon)
H. Sakai, S. Tabata, M. Kimura, S. Yabe, Y. Isa, Y. Kai, F. Sato, T. Yumoto, K. Miyano, M. Narita and Y. Uezono, "Active Ingredients of Hange-shashin-to, Baicalelin and 6-Gingerol, Inhibit 5-Fluorouracil-Induced Upregulation of CXCL1 in the Colon to Attenuate Diarrhea Development"Biol. Pharm.Bull.., 2017, 40, (12), 2134.
14) Cell
(SH-SY5Y cell)
R. Watanabe, T. Kurose, Y. Morishige and K. Fujimori, "Protective Effects of Fisetin Against 6-OHDA-Induced Apoptosis by Activation of PI3K-Akt Signaling in Human Neuroblastoma SH-SY5Y Cells."Neurochem. Res.., 2018, 43, (2), 488-499.
15) Cell
(MC3T3-E1[Derived from mouse cranial crown])
A. Oyane, M. Nakamura, I. Sakamaki, Y. Shimizu, S. Miyata and H. Miyaji, "Laser-assisted wet coating of calcium phosphate for surface-functionalization of PEEK"PLoS ONE ., 2018, doi:10.1371/journal.pone.0206524.
16) Cell 
CRC Cell [Colorectal cancer]、HT29 Cell[Adenocarcinoma of the human colon]
T. Fuji, Y. Umeda, A. Nyuya, F. Taniguchi, T. Kawai, K. Yasui, T. Toshima, K. Yoshida, T. Fujiwara, A. Goel and T.Nagasaka, "Detection of Circulating MicroRNAs with Ago2 Complexes to Monitor the Tumor Dynamics of Colorectal Cancer Patients during Chemotherapy."Int. J. Cancer., 2018, doi: 10.1002/ijc.31960 .
17) CEll
(SFXN2 knockout HEK293)
E. E. Mon, F. Y. Wei, R. N. R. Ahmad, T. Yamamoto, T. Moroishi and K. Tomizawa, "Regulation of mitochondrial iron homeostasis by siderofexin 2 "J Physiol Sci., 2018, doi:10.1007/s12576-018-0652-2.
18) Cell
(Human umbilical vein cell line [EA.Hy926])
Y. Xu, C. Huang, M. Liu, N. Chen, W. Chen, C. Yang, Y. Zhao, X. Li, J. Duan, S. Liu and S. Yang, "Survivin regulated by autophagy mediates hyperglycemia-induced vascular endothelial cell dysfunction"Exp. Cell Res.., 2018, doi: 10.1016/j.yexcr.2018.01.037.
19) Cell
(MIAPaCa-2[Human Pancreatic Cancer])
M. Akimoto, R. Maruyama, Y. Kawabata, Y. Tajima and K. Takenaga, "Antidiabetic adiponectin receptor agonist AdipoRon suppresses tumour growth of pancreatic cancer by inducing RIPK1/ERKdependent necroptosis"Cell Death Dis., 20189, 804.
20) Cell
(Fibro adipogenic progenitor cells:FAPs)
Y. Saito, T. Chikenji, T. Matsumura, M. Nakano and M. Fujimiya, "Exercise enhances skeletal muscle regeneration by promoting senescence in fibro-adipogenic progenitors"Nat. Commun.2020, doi:10.1038/s41467-020-14734-x.

Q & A

Q

Can I use filter other than 490 nm?

A

We recommend the 490 nm filter, but a difference of 490±2-3 nm is OK for measurement. 450 nm filters can also be used, however, we found that the absorbance value will be higher than that of 490 nm.

Q

Is there any correlation with Cell Counting Kit-8 in LDH cytotoxic assay?

A

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

Q

The LD50 value differs significantly from previous measurements. Is there anything I can do?

A

The LD50 value may be affected by the test substance depending on the condition of the cells. The LD50 value is affected by the condition of the cells.
The LD50 value can be obtained more reliably by using a narrower dilution factor when preparing a dilution series of the test substance.
It is recommended to set the concentration of the test substance to be used according to the following procedure.

General Protocol

(1) Repeat 10-fold dilution based on the highest concentration of the test substance available, and conduct toxicity testing over a wide sample concentration range of about 5-6 digits. [Figure ①]
(Using the operation in (1), establish a rough estimate of the highest predicted concentration in the concentration range at which toxicity will appear.
(ii) Based on the result of (i), 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 conduct toxicity testing in a concentration range covering 2-3 orders of magnitude. [Figure ②]
(iii) Repeat step (ii) to finally establish a concentration range where at least 3 sample concentrations can be plotted between 20-80% cytotoxicity. [Figure ③]

Q

Can I test LDH isozyme with this kit?

A

This kit is designed to measure total LDH activity, so it cannot measure LDH1, LDH2, LDH3, etc. separately.

Q

Can I store the sample for a long time of period?

A

It is possible to store the sample under 0-4℃ for one week1).
Store at room temperature2)may cause the LDH to lose activity, so it is not recommended.

<Reference>
(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 mammalian cells in perfusion reactors.", Biotechnol Bioeng. 1992; 39, (3), :320..

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

Q

Is this kit includes LDH standard?

A

This kit is generally for cytotoxicity assay, so the LDH standard in not included in this kit.

Q

Is it necessary to test immediately after adding Stop solution?

A

After adding the stop solution, please test the sample within 3 hours. In such cases, the plate should be protected from light.

Q

What can I do to decrease the variation between wells?

A

Please check the following possible factors.

① Bubbles on the medium surface

Remove bubbles by the following methods
Remove bubbles with the tip of a syringe.
(If you have a centrifuge for 96-well plates) Centrifuge at 1,000 x g for 1 minute.

②Reagent concentration is changing due to the evaporation during incubation

The outer wells of the microplate easily evaporate during incubation, so when incubating for a long time, do not use the outer wells for measurements.

③Large time difference between the first and last wells when adding reagents

We recommend using a multichannel pipette for Working Solution and Stop Solution, which is added to all wells.

④The added reagents are not well mixed

When Lysis Buffer, Working Solution, and Stop Solution are added, mix the added reagents and medium by mixing with a plate mixer or by lightly tapping the side of the plate with your finger.
Especially, please note that the amount of Lysis Buffer added is small and affects the value of high control.
When tapping the plate, make sure that the medium in the wells does not leak out.

⑤The (multichannel) pipette used is not accurate

Make sure that the pipette weighing is accurate.

Q

What can I do to decrease the background?

A

The following are possible causes
(1) LDH in serum in the medium raises the background. Use serum-free medium or medium prepared with a serum content of 5% or less.
(2) Strongly reducing substances in the test substance or medium may cause the dye WST to false-achromatize and raise the background. Please confirm this by measuring a reagent blank.
(In addition, common media such as DMEM, RPMI, F-12, etc. usually do not contain reducing substances).

Q

When I trying to optimize the cell numbers, there is no difference between high and low controls?

A

①The absorbance of the low control may be high due to the high background. Refer to the followings,

LDH in serum in the medium raises the background. Use serum-free medium or medium prepared with less than 5% serum.
If the test sample or culture medium contains strongly reducing substances, the dye WST will false-chromatize and raise the background. Please confirm this by measuring a reagent blank.

②Possible residual life cells in high control。

To ensure that the Lysis Buffer is evenly distributed throughout the wells and that all cells are dead, shake the plate gently after adding the Lysis Buffer to ensure that the Lysis Buffer is thoroughly mixed.

Handling and storage condition

Handling and storage condition
0-5°C, Protect from light
Danger / harmful
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