Overview

  • Product name
    LDH Assay Kit (Cytotoxicity)
    See all Lactate Dehydrogenase kits
  • Detection method
    Colorimetric
  • Sample type
    Adherent cells, Suspension cells
  • Assay type
    Enzyme activity (quantitative)
  • Assay time
    1h 00m
  • Product overview

    LDH Assay Kit (Cytotoxicity) ab65393 (previously called LDH-Cytotoxicity Assay Kit II) uses WST reagent for a fast and more sensitive detection of LDH released from damaged cells.


    The LDH assay protocol uses an enzymatic coupling reaction: LDH oxidizes lactate to generate NADH, which then reacts with WST to generate a yellow color. The intensity of the generated color correlates directly with the cell number lysed. 


    As WST is brighter than other cell viability reagents, less culture medium is required for the assay, and thus the background from serum and culture medium is significantly reduced. Cells can be cultured in regular 10% serum containing medium; no reducing serum or special medium is required for the assay.


    In addition, since WST is very stable, the reaction can be read multiple times and can be stopped at any time point during the reaction.


    LDH activity can be easily quantified by spectrophotometer or plate reader at OD450nm.


    LDH assay protocol summary:
    - transfer culture medium into new plate
    - add LDH reaction mix and incubate for 30 min at room temp
    - analyze with microplate reader

  • Notes

    If you would like to use a fluorometric reading, please refer to LDH-Cytotoxicity Assay Kit (Fluorometric) (ab197004)

    This kit is more sensitive than our colorimetric LDH Cytoxicity Assay Kit ab65391.

    A cell death assay / cytotoxicity assay classically assesses the level of plasma membrane damage to a cell population. Lactate dehydrogenase (LDH) is a stable enzyme, present in all cell types, which is rapidly released into the cell culture medium upon damage of the plasma membrane. LDH is the most widely used marker used to run a cytotoxicity assay.

    Review our cell health assay guide to learn about our other kits to perform a cell viability assay, cytotoxicity assay or cell proliferation assay.

     

  • Platform
    Microplate reader

Properties

Images

  • Bafilomycin A1 (BafA1) toxicity was assessed in Human hepatic (Huh7.5) cells after 24 hours at different concentrations (12.5nm, 25nm, 50nm and 100nm) were administered to cells using LDH cytotoxicity assay kit (ab65393). Cytotoxicity was measured by subtracting LDH content in remaining viable cells from total LDH in untreated controls. 

  • Jurkat T cells were cultured in 96-well plate in 100 μl of culture medium. LDH Assay was performed using 10μl of culture medium using the WST probe. Low control (white bar); High control (black bar).

  • Comparison of WST-1 and INT based LDH assays. 3T3 cells were cultured in a 96-well plate in 100 µl of culture medium. The LDH assay was performed using 10 µl of culture medium using WST-1 (Brown bar) and INT (Green bar) methods. The WST-1 based LDH assay is more stable and sensitive than the INT based method.

Protocols

References

This product has been referenced in:
  • Jackson TC  et al. BrainPhys® increases neurofilament levels in CNS cultures, and facilitates investigation of axonal damage after a mechanical stretch-injury in vitro. Exp Neurol 300:232-246 (2018). Functional Studies . Read more (PubMed: 29199132) »
  • Smith JA  et al. RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity. Mol Ther Nucleic Acids 10:103-121 (2018). Functional Studies . Read more (PubMed: 29499926) »
See all 35 Publications for this product

Customer reviews and Q&As

1-10 of 53 Abreviews or Q&A

Question
Answer

The kit ab102526 is more suitable for measuring the LDH activity in tissue culture supernatants - It can also be used for tissue extracts.

ab65393 is only tested with cells and cell extracts - tissue extracts are yet to be tested therefore I we cannot confirm its specificity. More likely it will work with tissue extracts as well. Please note you might need to optimize the amount of tissue by trying several dilutions.

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Answer

Yes, the lab suggested adding 100ul of the media to be tested to 100ul of the LDH reaction mix then read optically. You can make/use a standard curve if you know the conc of the LDH you use for it. We have not provided the conc of LDH in our kit due to proprietary restrictions.

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Question
Answer

Thank you for your inquiry and your patience with this reply.

I've asked the supplying lab for more information about the components of the stop solution, but they were unable to disclose what they use since they consider it "proprietary information." I appreciate that this is frustrating. The best I can do is try to get ithe stop solution added as a separate component on our catalogue, but unfortunately you would have to purchase it from the lab. If you would just like to purchase an entire kit instead, I can offer you a 15% discount.

Please let me know how you would like to proceed. I look forward to your reply.

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Answer

Thank you for contacting us.

The cell lysis solution should be used to prepare the High control only. Please use 10% of cell lysis solution i.e. for 100 ul use 10 ul cell lysis solution.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

I have heard back from the lab with the following information:

There are numerous differences between these two assay kits. For details, I have attached the datasheets, but here are the main differences:






Kit
ab65391
ab65393

Detection wavelength
500 nm
450 nm

Principle
LDH activity can be determined by a
coupled enzymatic reaction: LDH oxidizes lactate to pyruvate which then reacts with
tetrazolium salt INT to form formazan. The increase in the amount of formazan produced in
culture supernatant directly correlates to the increase in the number of lysed cells.
The assay utilizing an enzymatic coupling reaction: LDH oxidizes
lactate to generate NADH, which then reacts with WST to generate yellow color. The intensity
of the generated color correlates directly with the cell number lysed.

Special advantages:

Since WST is brighter,
less amount of culture medium is required for the assay, and thus the background from serum
and culture medium is significantly reduced. Using the assay, cells can be cultured in regular
10% serum containing medium, no reducing serum or special medium is required for the
assay. In addition, since the WST is more stable, the reaction can be read multiple times, and
can also be stopped at any time point during the reaction. LDH activity can be easily quantified
by spectrophotometer or plate reader at OD450 nm. The kit provides all necessary reagents
including LDH positive control.






One can use only the cell media with these kits.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question
Answer

Thank you for contacting us.

Can you please clarify the difference between your CON samples and your LC? It seems like both sets of samples are cells without treatment, is that correct?

What sort of background readings were you getting on your plate?

I would suggest checking your cells under a microscope to validate that the cells in your low control are healthy and intact, and that the cells in your high control have been effectively lysed. Because the OD readings in your high control are not quite at the ˜2.0 OD level that is recommended in the protocol, the lysis may not be effective, or you may need to seed more cells per well to get accurate readings.

Once the low and high controls are optimized, a negative reading would indicate that you have more cells in your treated samples than in your controls. If the wells are all seeded evenly, this could indicate that your treatments may be encouraging cell division.

I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting us.

This kit is compatible with cells. Therefore I just want to make sure that you are planning on using this assay with the BAL cells as well.

In that case you may have to incubate the BAL fluid with the cell lysis solution, so all the cells in the lavage break down and release any LDH in the fluid. Ideally you may want to pellet down the cells and use them directly for the low control as well.

I hope this information has been useful for you. Please let me know if you have any other questions

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Answer

Thank you for contacting us.

We haven't tested this kit with plant material yet so I am sorry we are not aware of any interference of photochemicals with the absorbance reaction of this kit. This kit uses WST1 compound which has been successfully used in assaying LDH cytotoxicity with different plant materials; the information is published in many research paper. Theoretically, I can say the phytochemicals should not be having any effect on absorbance.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you very much for letting me know. I am pleased to know the kit is now working well for you.
If you have any questions or concerns in the future, please do not hesitate to contact us again.
Until then, I wish you all the best with your research.

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1-10 of 53 Abreviews or Q&A

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