LDH Colorimetry Cytotoxicity Assay

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.

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.

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 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 achieve a final concentrationof 1x, for example,100μLof culture medium with10μLof lysis buffer(10×)followed by45min (i.e., simultaneously with the samples)Collected 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 the experimental readings. The kit includes one tubeLDH (500x)positive control, which can be diluted withPBS+1% BSAto make a500xdilution as the positive control for the experiment.

The following example describes a compound cytotoxicity assay using a96well cell plate.

3.1Cell treatment

1)In a96-well cell culture plate, seed the experimental cells at an appropriate density, with100-200μLof culture medium per well. Serum, such asFBSmay containLDH. If this background is high enough to affect the experimental results, it is recommended to use56℃,0.5 hoursto inactivate theFBSwhen preparing the complete culture medium for seeding the cells.

2)Perform compound treatment on the cells according to experimental requirements. Each experimental plate should includea toxic reference compound control, a solvent vehicle control, and acell-free complete medium control.

3)According to experimental requirements, samples are taken at a certain time after cell treatment forLDHdetection.

3.2 LDHdetection.

1)Take50 μLof the above-mentioned culture medium sample and add it to a completely transparent96-well plate. The maximum detectable value should preferably not exceed3when reading the plate at15 min. If it exceeds3, it is recommended to appropriately dilute the sample before detection.

2)Calculatethe amount of LDHdetection reagent needed:For 96-well plate detection, calculate based on adding50µLof sample with50µL of LDHdetection reagent.For 384-well plate detection, calculate based on adding12.5µLof sample with12.5µL of LDHdetection reagent.

3)Remove the LDHdetection reagent from storage, thaw it, and equilibrate it to room temperature(22℃-25℃).Invert thoroughly to mix well. After taking the required amount of reagent according to experimental needs, use it immediately; store any remaining reagent by freezing at-20℃or lower.

4)Add to each well50 µLAdd sample50 µL LDHdetection reagent. When handling multiple samples, use a multichannel pipette to add the detection reagent to ensure consistent incubation time.

5)Shake the plate at medium speedfor 15 secto mix thoroughly, then incubate in the dark at room temperature.After15 minincubation,read the absorbance at 490 nm. For low-concentrationLDHassays, the incubation time can be extended to30 minto improve detection sensitivity.

6)AddLDHdetection reagentand incubate for15 min(or30 min).After incubation, add50µLof stop solution(1N HCl),shake the plate at medium speedfor 15 secto mix well.Read the absorbance at490 nmwithin 1 hour; the signal remains essentially stable. The stop solution is1N HCland should be handled with care and proper protective measures.

It is not recommended to mix different batches.