LDH Cytotoxicity Assay Kit

 

Product Name	Cat. No	Spec.
LDH Cytotoxicity Assay Kit	G1610-100T	100T

 

 

Lactate dehydrogenase (LDH) is a terminal enzyme of the glycolytic pathway, widely found in animals, plants, and microorganisms, and is involved in catalyzing the reversible reaction between pyruvate and lactate. Normally LDH is abundant in the cytoplasm of cells, and normal cells cannot pass intracellular LDH freely through the cell membrane due to the barrier protection of the cell membrane. However, when cells are damaged or die, the barrier protection of the cell membrane disappears and intracellular LDH is released into the culture medium. Cytotoxicity can be quantified by detecting the amount of LDH released into the culture medium by cell membrane rupture.

 

The main principle of Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit is to use LDH to reduce NAD+ to NADH, and further NADH and INT (tetrazolium salt) catalyzed by Diaphorase (Lipoic Acid Dehydrogenase) to produce NAD+ and red filth, the amount of filth produced and the amount of LDH is a linear correlation, and is able to produce an absorption peak at 490 nm. Absorption peak at 490 nm, so it can be quantitatively analyzed by the instrument. This kit is mainly used for cytotoxicity assays based on LDH release as an indicator, but can also be used for cell proliferation and toxicity assays based on total cellular LDH.

 

 

Ship with wet ice; Store at -20℃ for up to 12 months. Avoid repeated freeze-thaws of Enzyme Solution. INT Solution should be protected from light.

 

 

Component Number	Component	G1610-100T
G1610-1	10× Cell Lysis Buffer	1.5 mL
G1610-2	Reaction Substrate	2×1 mL
G1610-3	INT Solution	200 μL
G1610-4	Enzyme Solution	2×150 μL
G1610-5	Reaction Buffer	10 mL
Manual		One copy

 

 

1. Sample Preparation:

1) Detection of extracellular LDH

a. Cell seeding: Seed cells in 96-well cell culture plate for appropriate time depending on the type of cells. The confluence of cells to be tested should exceed 80-90%. And multiple controls can be set according to experimental requirements.

background control：contains culture medium only and no cells.

sample control：contains culture medium and cells. Untreated cell wells.

Sample maximum enzyme activity control: contains culture medium and cells. Detection of total intracellular LDH after lysis of untreated cells

Treated Sample (experimental group): According to the desired method, treat cells with appropriate drug at different concentrations.

b. Cell treatment: Remove the culture medium and wash cells once with PBS solution (recommended G4202). Add fresh culture medium (culture medium containing low serum, serum-free or drug-containing culture medium) into each well, incubate for a certain period.

c. Sample collection: Centrifuge the cell culture plate at 1,000×g for 5 min. Transfer 80 μL of supernatant from each well into a new 96-well plate, after which the samples were assayed sample preparation for the control well with the maximum enzyme activity of the sample, please Refer to the sample preparation steps for total intracellular LDH assay).

2) Detection of total intracellular LDH

a. Cell seeding: Cells are inoculated in 96-well cell culture plates at a suitable density, and multiple subgroups can be set up according to experimental needs;

b. Cell treatment: according to the experimental design, use drug-containing or drug-free medium (may contain serum) to incubate cells;

c. Cell lysis: After reaching the predetermined time, remove the original medium, wash it with PBS once, add 120 μL of cell lysis working solution (10× cell lysis solution diluted 10 times with PBS), and incubate in the incubator for 30-60 min ;

d. Sample collection: Centrifuge the cell culture plate at 1,000× g for 5 min, and add 80 μL of the lysis supernatant to a new 96-well plate, followed by assay.

3) (Optional) LDH standard sample preparation

If absolute quantification of LDH enzyme activity is desired, purchase LDH standard individually and prepare LDH standards at different concentrations: 10 mU/mL, 5 mU/mL, 2.5mU/mL, 1.25 mU/mL, 0.65 mU/mL and 0 mU/mL. At 80 μL per well, the gradient was added to the 96-well plate.

2. LDH Detection:

1) According to the number of samples to be tested, refer to the table below to prepare an appropriate amount of LDH detection working solution.

Note: it corresponds to 96-well plate, and for other specifications of well plates can be adjusted as needed);

 

	1 Sample	10 Samples	20 Samples	50 Samples
Reaction Substrate	20 μL	200 μL	400 μL	1 mL
INT Solution	2 μL	20 μL	40 μL	100 μL
Enzyme Solution	3 μL	30 μL	60 μL	150 μL
Reaction Buffer	55 μL	550 μL	1.1 mL	2.75 mL
Total Volume	80 μL	800 μL	1.6 mL	4 mL

 

2) Add 80 μL LDH working solution into 80μL supernatant in 96-well plate prepared in step 1 (Vsample：VLDH detection working solution = 1:1), mix by gentle tapping.

3) The cells were incubated in a cell culture incubator, protected from light for 30 min, or wrapped in tin foil and incubated on a horizontal shaker for 30 min at room temperature;

4) Measure the absorbance of all controls and samples with a plate reader at 490nm. The reference wavelength should be 600nm or greater.

3. LDH Result Calculation:

1) Routine LDH (release) cytotoxicity assay:

a. Subtract the OD490 of the background control from the OD490 of the sample control, sample maximum enzyme activity control, treated sample wells, etc., and use them for subsequent calculations.

b. Calculation of cytotoxicity or death rate (%) = (OD490 of treated sample - OD490 of sample control) / (OD490 of maximum enzyme activity of sample control - OD490 of sample control) × 100%

2) Relative quantification of LDH enzyme activity (according to the calculation results, it is possible to compare whether there are statistical differences between different sample treatment groups, etc.):

a. Determine the OD490 of a known concentration of LDH standard;

b. LDH activity of the sample to be tested (mU/mL) = (OD490 of sample well - OD490 of background blank control well) / (OD490 of standard product - OD490 of background blank control well) × standard concentration (mU/mL)

3) Absolute quantification of LDH enzymatic activity:

a. Measure the OD490 of a series of LDH gradient standard products, draw a standard curve according to the obtained absorbance value, and calculate the trend formula: Y(OD490) = A×LDH activity unit (mU)+B, the trend can be calculated by software such as Excel the slope and intercept of the line;

b. LDH activity in the detection system (mU) = (sample well OD490-background blank control well OD490-B)/A

c. Sample LDH activity (mU/mL) = LDH activity in the detection system (mU) / detection volume (mL)

 

 

1. The density of the cells should not exceed 85% or more, factors such as the state of the cells and the density of the cells will have some effect on the cellular LDH release, and the samples are prepared, try to complete the test on the same day, do not freeze.

2. Serum contains lactate dehydrogenase, it is recommended to use serum-free or low serum medium, if you have to use 10% serum, please set up a cell-free control group to eliminate background interference.

3. The operation is as gentle as possible and bubbles are to be avoided so as not to affect the experimental results.

4. For your safty and health, please wear safety glasses, gloves, or protective clothing.

 

For Research Use Only!