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Product Details
| Cat # +Size | K313-500 |
|---|---|
| Size | 500 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Mammalian |
| Applications | This assay provides a convenient and Wst-based non-radioactive assay for measurement of activity of lactate dehydrogenase (LDH) which is a stable enzyme normally found in the cytosol of all cells but rapidly releases into the supernatant upon damage of plasma membrane. |
| Features & Benefits | • Simple one-step procedure; takes around than 1 hour • Fast and convenient |
| Kit Components | • WST Substrate Mix • LDH Assay Buffer • Cell Lysis Solution • Stop Solution • LDH (0.1 µg/ml) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Cell death or cytotoxicity is classically evaluated by the quantification of plasma membrane damage. Lactate dehydragenase (LDH) is a stable enzyme, present in all cell types, and rapidly released into the cell culture medium upon damage of the plasma membrane. LDH, therefore, is the most widely used marker in cytotoxicity study. BioVision’s LDH Cytotoxicity Assay Kit II utilizes the advanced WST reagent for a fast and more sensitive detection of LDH released from damaged 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. 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. Assay takes less than 1 hour.
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We would like to know if this kit can be used for supernatant samples that have been frozen?
It is best to use this with fresh samples rather than frozen. The protocol will have to be changed slightly to accommodate the frozen samples. Also, the success of the assay would depend on numerous extra criteria including how well the samples were frozen, how long they were frozen, number of times they were thawed out etc, which increases the complexity. Therefore I would recommend you to stick with fresh samples.
1. I have a customer who has the following questions in regards to you kit K313-500.The cells are primarily cultured chick embryonic forebrain neurons co-grown with glial cells. She just needs to know whether her culture is healthy in general along their life time in culture. Would you please advise whether your kit is appropriate for my purpose?
2. If, yes, how?
3. the plating density of the cells is fixed at 3000 cells/mm^2. Does she have to plate the cells using a 96-well plate? May she use 24-well, 12-well plate, and a microelectrode array chamber, then at day 7,14,21, and 28, she could take out some supernatant to a 96-well plate to assay?
4. if this is ok, may I store the supernatants taken out on different days, store them at -20C or -80C, then perform the LDH assay at one time?
2. If, yes, how?
3. the plating density of the cells is fixed at 3000 cells/mm^2. Does she have to plate the cells using a 96-well plate? May she use 24-well, 12-well plate, and a microelectrode array chamber, then at day 7,14,21, and 28, she could take out some supernatant to a 96-well plate to assay?
4. if this is ok, may I store the supernatants taken out on different days, store them at -20C or -80C, then perform the LDH assay at one time?
Yes, this kit would be appropriate for this assay and I have explained below how. For this particular kit, the client can actually look at the LDH released into the cell media in her 7-28 day cultures which I assume would be very low relative to the high controls, which would indicate that the cells are viable. Also, by doing a comparative LDH level analysis between the different time points the client would be able to see by which day/time the cells release lowest LDH and have highest viability.I would not store samples. Just do a fresh testing at the different day time points.The client can grow cells in any size plate, but would need to have them in a 96 well plate for the expt. If she wants to take just the sup, she can, but then all the controls also have to be taken appropriately. For eg, for the high cont., taking the sup and treating with cell lysis solution is not going to do the trick. She needs the cells for that.The number of cells for each assay will also need to be optimized as mentioned in the protocol.
We have a customer who is having a little difficulty using the LDH-Cytotoxicity Assay Kit II. The positive control is yielding an OD of 1.5 but the low and high control are both showing an OD of 0.6. Please could you advise as to the likely cause of this result.
To begin with, the client is using very few cells. We recommend the cell number to be within the range of 2-8 x10^4. Please request the client to increase their cell number. The amount of cells to be used per well depends on the cell types. To optimize the assay, you can do a quick testing by using 2, 4, 8 x 10^4 cells per well, and then follow the assay protocol to determine the cell number you should use. The high control should be OD450nm ~ 2.0 after 30 min treatment with 10 % Cell Lysis Solution, while the low control should be OD450nm < 0.8. The reaction time should be set at ~ 30 min. Once this is done, their issue with the ODs of HC vs LC will be resolved.They will need to optimize the dose and incubation time with the drug, to ensure that they get efficient results. Additionally they might have to do a solvent control also if they do not use PBS with their drugs.At what OD did they read the plate? Also, The reaction time can be decreased or increased depend on the color development. The plate can be read at multiple time points until the desired reading is observed. The high control should be OD450nm ~ 2.0, while the low control should be OD450nm < 0.8.
Might this assay be negatively effected by pan hematin or SnCl2? These are present in my samples.
Since this is a relative assay with the inclusion of the high and low controls, which will have the same pan hematin or SnCl2, the final result will not be affected by these. So it should be alright to use these samples with this kit.
I wanted to ask you about your product (LDH-Cytotoxicity Assay Kit II (500assay) ). Is it suitable to be used for both human and mouse cell culture?? Also regarding measuring the absorbance is it ok if the reference wavelength is 620 nm or should it be 650 nm.
Yes, both mouse and human cells can be used with this kit. This kit is in fact compatible with all mammalian samples. The reference wavelength for detection for this assay is 450 nm, not 650 nm. You can use a slightly different wavelength than the recommended one as long as your plate reader specifications mention that range of band width. For example if the band width range is +/- 20, you can use 430 - 470 nm wavelength.
In your opinion would the phenol red in a cell culture medium interfere with the reaction/reading of products K313-500
Yes, phenol red in the media can contribute to the final reading, which is why we suggest using the background control with just the cell culture media, which will account for any contribution by everything in the media including the phenol red.
What is the difference between K311 and K313?
There are numerous differences between these two assay kits. Detection wavelength - K311:500 nm, K313: 450 nmPrinciple - K311: 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. K313: 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 of K313: 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.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Zhibing Lin, A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model. Mol Ther Oncolytics, June 2019; 31384666.
Swechha M. Pokharel, Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response. Nat Commun., April 2019; 30931941.
Kim, BY et al., (2017) Synthetic secapin bee venom peptide exerts an anti-microbial effect but not a cytotoxic or inflammatory response, J Asia-Pacific Entomology, 2017, 20(1): 151-155
Qin J et al., (2017) Effect of mild hypothermia preconditioning against low temperature (4°C) induced rat liver cell injury in vitro, PLoS One, 2017, 12(4): epub
Lin, Sheng-Hsuan et al. (2016) Arecoline-induced pro-fibrotic proteins in LLC-PK1 cells are dependent on c-Jun N-terminal kinase, Toxicology. 2016 Feb 17;344-346:53-60.
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