LDH Colorimetry Cytotoxicity Assay
LDH Colorimetry Cytotoxicity Assay
Product Details
Product Details
Product Specification
| Stability & Storage | Dry ice transport. Upon receipt, store the LDH assay reagent and the LDH positive control protected from light at −80°C; store the stop solution (1N HCl) and the cell lysis buffer (10×) at −20°C or 4°C. Refer to the reagent vial label for the expiration date. After first use, it is recommended to aliquot the LDH assay reagent as needed and store it protected from light at −20°C or below; for long-term storage (>2 months), store at −80°C. The LDH assay reagent should not be subjected to more than three freeze–thaw cycles after initial use. For the LDH positive control, after first use, we recommend long-term storage by aliquoting as needed at −20°C or below, and short-term storage (up to 2 weeks) at 4°C. |
Background
The LDH Colorimetric Cytotoxicity Assay Kit determines the toxicity of external stimuli or compounds to cells by measuring the lactate dehydrogenase (LDH) released by cells. LDH is widely present in the cytoplasm and is released into the culture medium when cell membranes are damaged or ruptured. With a half-life of approximately 9 hours in the culture medium, extracellular LDH is often used as an indicator of cell health to reflect the toxic effects of external stimuli or compounds. The LDH colorimetric cytotoxicity assay detects LDH activity through an enzymatic reaction. Released LDH converts NAD to NADH, and a reductase then uses NADH to convert iodonitrotetrazolium violet (INT) into a red formazan product. The absorbance of the formazan product can be measured at 490 nm (or 492 nm) for quantification, with its amount being directly proportional to the LDH activity in the sample. The LDH colorimetric cytotoxicity assay kit features a high signal-to-noise ratio, good reproducibility, and excellent stability. Its ready-to-use homogeneous formulation requires only a single addition step, reducing experimental errors that may arise from multiple additions. Additionally, this reagent does not require cell lysis, allowing continuous testing on the same cell sample and further simplifying experimental design. The LDH colorimetric cytotoxicity assay kit can be used in combination with other assays for assessing cell health on the same experimental sample, such as the UA-Glo® CTG Cell Viability Assay Kit (Cat# UA070103), Caspase A cell apoptosis assay kit (Cat# UA079057) and the UA-Glo® fluorescent cell viability assay kit (Cat# UA079015) were used for multiplexed detection to better elucidate the mechanisms of cytotoxicity.
Components
LDHThe components and specifications of the colorimetric cytotoxicity assay kit are as follows. Detectable96/384-well plate reaction numbers are calculated based on adding50/12.5μLsample and50/12.5μLdetection reagent per well.
Specifications |
Components and specifications |
96-well plate reaction number |
384-well plate reaction number |
200T |
LDHdetection reagent:12 mL stop solution(1N HCl):12 mL cell lysis buffer(10×):1 ml LDHpositive control(500x): 50μL |
200 |
800 |
1000T |
LDHdetection reagent:12x5 mL stop solution(1N HCl):60 mL cell lysis buffer(10×):5 ml LDHpositive control(500x): 50 μL |
1,000 |
4,000 |
Protocol
Experimental Control: Under most experimental conditions, it is not recommended to directly compare the absolute readings of sample measurements. It is suggested to set up the same reference controls on each experimental plate; for example, in a tumor cell cytotoxicity assay, use the same toxic reference compound, solvent vehicle control, and medium blank control. Normalize the sample measurement values using the readings from the toxic reference compound and solvent vehicle control before data processing and comparison.LDHCytotoxicity assays using the colorimetric LDH method can calculate cytotoxicity according to the following formula: (%): Cytotoxicity(%) = 100x (Sample measurement value-Solvent vehicle control measurement value)/(MaximumLDHrelease control measurement value-Solvent vehicle control measurement value). The maximumLDHrelease control can be either the toxic reference compound control or, prior to the assay,45minbefore adding the solvent vehicle control, add lysis buffer(10×)to a final concentrationof 1x, for example,100μLof culture medium with10μLof lysis buffer(10×)followed by45min (i.e., simultaneously with the samples)Collect the supernatant. It is recommended to set up a cell-free complete medium well as a background control for detection; this background reading can be used to correct experimental readings. The kit includes one tube.LDH (500x)Positive control, which can be diluted withPBS + 1% BSAto a500xconcentration and used as the positive control in the experiment.
Reagent preparation and storage (Reference:Storage conditions):
1.LDHAssay reagent: After thawing upon first use, equilibrate to room temperature(22℃-25℃)and mix thoroughly by inversion. Take the required amount according to experimental needs and use immediately; freeze any remaining reagent immediately and store at-20℃or below.
2.Cell lysis buffer(10×): After thawing upon first use, equilibrate to room temperature and mix thoroughly by inversion; it can be stored at4℃
3.Stop solution(1N HCl): After thawing upon first use, equilibrate to room temperature and mix thoroughly by inversion; it can be stored at4℃
4.LDHPositive control(500x): After first use, aliquot as needed and store at-20℃or below.
The following example is for a compound cytotoxicity assay using a96-well cell plate.
3.1Cell handling
1)Seed experimental cells at an appropriate density into a96-well cell culture plate, with100–200μLof medium per well. Serum such asFBSmay containLDH; if this background is high enough to affect the experimental results, it is recommended to prepare the complete medium for seeding cells using56℃,0.5-hour inactivatedFBS
2)Perform compound treatment on the cells according to experimental requirements. Each experimental plate should include atoxicity reference compound control, a solvent vehicle control, and ano-cell complete medium control.
3)At a specified time after cell treatment, take samples forLDHdetection as required by the experiment.
3.2 LDHdetection
1)Take50 μLof the above experimental medium sample and add it to a completely transparent96-well experimental plate. The maximum detection value is within15 minutes.It is best not to exceed when reading the board.3. If it exceeds3It is recommended to dilute the sample appropriately before testing.
2)CalculationLDHTest reagent usage:96-Inspection of the Kong experimental board according toFiftymicroliterSample addedFiftyµL LDHRequired for test reagent calculation,384-Inspection of the Kong experimental board according to12.5µLSample added12.5µL LDHRequired for test reagent calculation
3)Take out LDHTest reagent, melt and equilibrate to room temperature.(22°C-25°C), invert and mix thoroughly. After taking the required amount of the test reagent according to experimental needs, use it immediately; any remaining reagent should be immediately frozen and stored at-20°CSave below and including this level
4)To each hole50 µLSample addedFifty µL LDHDetection reagent. When handling multiple samples, use a multichannel pipette to add the detection reagent to ensure consistent incubation times.
5)Medium-speed vibration plate15 secondsMix well and incubate in the dark at room temperature.After 15 minutes,measure the absorbance at490 nm.For low-concentrationLDHassays, the incubation time can be extended to30 minutesto improve detection sensitivity.
6)Add theLDHdetection reagent,incubate for15 minutes (or30 minutes),then add 50 µLof the stop solution(1N HCl),shake the plate at medium speed for15 secondsto mix thoroughly.Read the absorbance at490 nm within1 hour;the signal remains essentially stable. The stop solution is1N HCl,so handle and protect yourself carefully when using it.
Guidelines
Do not mix different batches.
Do not alter the volume of the test reagent without rigorous validation.
For research use only.
