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LDH-Cytotoxicity Colorimetric Assay Kit

based on 59 citations in multiple journalsLDH-Cytotoxicity Colorimetric Assay Kit594.7 5
Catalog #: K311

In stock

$265.00

Product Details

Cat # +Size K311-400
Size 400 assays
Detection Method Absorbance (500 nm)
Species Reactivity Mammalian
Applications This assay provides a convenient and non-radioactive alternative to the [3H]-thymidine and the [51Cr] release assays The assay is based on 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 less than 1 hour
• Fast, sensitive and convenient
Kit Components • Catalyst, Lyophilized
• Dye Solution
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. The LDH-Cytotoxicity Assay Kit provides a fast and simple method for quantitating cytotoxicity based on the measurement of activity of lactate dehydrogenase (LDH) released from damaged cells. Unlike many other cytoplasmic enzymes which exist in many cells either in low amount (e.g., alkaline and acid phosphatase) or unstable, LDH is a stable cytoplasmic enzyme present in all cells and rapidly released into the cell culture supernatant upon damage of the plasma membrane. 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 formazan dye is water-soluble and can be detected by spectrophotometer at 500 nm. The LDH-cytotoxicity assay is sensitive, convenient, and precise, and is applicable to a variety of cytotoxicity studies. Assay takes ~0.5–1 hr.


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Can either the cell viability assay and LDH-Cytotoxicity Assay Kits be used to measure the inhibition of lysis instead of direct lysis?
The kit K254 measures cell viability by monitoring ATP levels; cells are lysed in the Nucleotide releasing buffer and the ATP levels are then analyzed. The kit K311 measures the cell death by monitoring the LDH levels in the cell culture supernatant. “LDH is a stable cytoplasmic enzyme present in all cells and rapidly released into the cell culture supernatant upon damage of the plasma membrane.” I think that the kit K311 will be more appropriate in your case.
Is it OK to use 540 nm filter for reading?
540 nm can read ~50% signal, so as long as you have good readings, it’s fine.
Can medium be stored at –20C before assay, for how long?
Yes, medium can be stored frozen for at least 2 months.
Can reagents be stored at –20C after reconstitution?
The reconstituted Catalyst solution is not stable under frozen conditions
What kinds of readings are expected for low and high controls?
Low control: OD 0.5 – 1.0 and High control: OD 2-3.
What is the Sensitivity Limit?
0.05 U/ml.
Does phenol red affects the assay?
Phenol red will increase the OD reading (0.1-0.2). However, if your background, control media and testing media all contain the same amount of phenol red, the affect can be ignored, since it will be subtracted from your testing samples.
Does serum affects the assay?
Serum will increase the OD reading (0.5). However, if your background, control media and testing media all contain the same amount of serum, the affect might be ignored (depending on each situation), since it will be subtracted from your testing samples.
I got same value in high and control samples. Why?
The high control is treated with 1% Triton X100, thus cells will release LDH into media, whereas low control only control regular medium (not much LDH will be released). The differences should be significant. There are several possibilities that I can think of:
1) Maybe the cells do not contain LDH;
2) Maybe the Triton X-100 concentration was not correct, or the low control cells were also died. Generally, low control should have an OD reading 0.5-1, whereas high control has an OD reading 2-3.
3) The machine filter setting might not be correct.
Why do I have a high background? What can I do to lower it?
The high background could be attributed to two factors: The culture medium contains high LDH, probably from the serum.The high LDH could be from leaky cells. The remedy could be: Reduce the serum concentration to 0.5%. Since there is always some cells that will leak LDH, reduce the number of cells in the assay. By decreasing the serum concentration or the number of cells or even both you will lower the control reading.
Is a stop reaction solution necessary? When will it be OK not to use it?
If you use a microtiter plate, one plate can normally be read within 1 minute. During the time, the signal would not change significantly. However, if you have multiple plates and the samples will be read over a 3-minutes period, you may consider using another LDH assay kit (K313-500) in which a stop solution is provided
Does pyruvate present in the DMEM medium interfere with LDH reaction?
We used excessive amount of lactate (~100mM) in the reaction system, which will promote LDH catalyzed conversion of lactate to pyruvate, and inhibit the conversion of pyruvate to lactate. So the pyruvate in the medium should not affect the assay.
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.
Does 15 % FBS in your culture media interfere with the assay?
15% FBS is a lot of serum and this can interfere because of the presence of proteins and enzymes. We recommend using 1% serum in the medium for this assay.
Can you freeze the culture media before testing?
We do not recommend freezing the culture media before the assay for best results. If absolutely necessary the media can be collected into tubes, spun down to remove any cell debris/floating material and then stored in aliquots at -80C. Media frozen right after collection and without any freeze-thaw cycles are still ok. But LDH activity will decrease on long storage.
Shuai, Yi et al. (2017) Characterization of Microcystin-Induced Dualistic Toxic Effects on Primary Rat Hepatocytes, J Environ Pathol Toxicol Oncol. 2017;36(1):15-27.
Long, Chengmei et al. (2016) Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant, anti‑inflammatory, and anti‑apoptotic activities, Mol Med Rep. 2016 Jun;13(6):4697-704.
Yang, Weihua et al. (2016) Antitumor effect of a polysaccharide isolated from Phellinus pullus as an immunostimulant, Biomed Rep. 2016 Mar;4(3):361-364.
Bucki et al., Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection. Antimicrob. Agents Chemother., Oct 2015; 59: 6274 - 6282.
Smith et al., Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures. Med Mycol, Sep 2015; 53: 645 - 655.
For more citations of this product click here
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