Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (565nm)
Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (565nm)
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| Usage | I. Instruments, consumables, and reagents required: 1. Microplate reader (capable of measuring absorbance at 565 nm) and CO2 incubator. 2. 96-well plates with transparent flat bottoms. 3. Centrifuge (optional). 4. Adjustable pipette and tips. 5. Deionized water. II. Reagent Preparation: Assay Buffer: Ready-to-use; equilibrate to room temperature before use; store at 4°C. Lactic Acid Solution: Ready-to-use; equilibrate to room temperature before use; store at 4°C. MTT Solution: Ready-to-use; keep on ice and protected from light throughout the experiment; aliquot and store at -20°C. PES Solution: Ready-to-use; keep on ice and protected from light throughout the experiment; aliquot and store at -20°C. LDH Positive Control: Ready-to-use; keep on ice and protected from light throughout the experiment; store in aliquots at -20°C. NAD+ Solution: Ready-to-use; keep on ice and protected from light throughout the experiment; store in aliquots at -20°C. Triton X-100 (10%): Ready-to-use; equilibrate to room temperature before use; store at 4°C. LDH Reaction Solution: Prepare 10 mL of LDH reaction solution, sufficient for one 96-well plate, by mixing 3.7 mL Assay Buffer, 1.4 mL MTT Solution, 800 μL NAD+ Solution, 100 μL PES Solution, and 4.0 mL Lactic Acid Solution. Prepare freshly for use. III. Experimental steps: 1. Determine whether the drug is suitable for this kit Some drugs may interfere with the reaction system and are therefore not suitable for this kit. Therefore, we recommend performing an initial pilot experiment to determine whether the target drug you plan to use is suitable for this kit. If the pilot experiment shows that the drug has an inhibitory effect, contact Absin technical support. (1) Culture the cells for more than 24 hours, centrifuge to remove the particles, and obtain the cell culture supernatant. (2) Add the cell culture supernatant to a 96-well cell culture plate at 200 μL/well, with 6 replicates. (3) Add 20 μL of the target drug to 3 wells, and add 20 μL of Assay Buffer to the other 3 wells as control wells. (4) Transfer 100 μL of each well to a new 96-well assay plate. (5) Add 100 μL of LDH Reaction Solution to each well. (6) Incubate the plate at 37°C for up to 30 min. (7) Read the absorbance at 565 nm using a microplate reader. (8) Evaluate the absorbance of the drug-treated wells and the control wells. If the drug-treated wells are significantly smaller than the control wells, the drug is not suitable for this kit. 2. Sample Assay (1) Determine the optimal concentration based on cell size and growth rate. Inoculate 200 μL of cells into a 96-well cell culture plate so that the cell density does not exceed 80-90% at the time of testing. (2) Aspirate the growth medium from the 96-well plate. Wash the cells once with PBS, then replace the growth medium with low-serum medium containing 1% serum and incubate for another 1 h. (3) Add 200 μL of 1% low-serum medium (without cells) to three wells as background controls and three wells as LDH positive controls (optional). (4) Induce cytotoxicity by the desired method and add 20 μL of drug to the appropriate wells in triplicate. Add 20 μL of Assay Buffer to three wells containing cells as spontaneous release, and add 20 μL of Assay Buffer to three wells containing only 1% low-serum medium without cells as background control. (5) Incubate the plate in a CO2 incubator at 37°C for the desired experimental time. (6) One hour before the scheduled assay, add 20 μL of 10% Triton X-100 solution to three wells containing cells as maximum release, and add 20 μL of LDH Positive Control to three wells containing only 1% low-serum medium without cells as positive control. Mix by pipetting several times and continue incubating in the CO2 incubator. (7) Centrifuge the 96-well tissue culture plate at 400 g for 5 minutes (optional, but recommended). (8) Transfer 100 μL of cell supernatant to a new 96-well assay plate. (9) Add 100 μL of LDH Reaction Solution to each well. (10) Incubate the plate at 37°C for up to 30 minutes. (11) Read the absorbance at 565 nm using a microplate reader. (12) Subtract background A565 levels from all wells. Note: The drug concentration added should be the final concentration in the reaction. The results of each experiment are calculated as "percent cytotoxicity," or the percentage of the total amount of LDH contained in the target cells. Therefore, for each experiment, there must be a set of control wells in which all target cells are killed using the 10% Triton X-100 solution provided in the kit. These are the "maximum release levels." Likewise, in every experiment, there must be a set of control wells in which no cytotoxic agent or cytotoxic cells are added, resulting in the lowest (spontaneous) LDH release. These are the "spontaneous release" wells. Cells treated with cytotoxic agents will release an amount of LDH that lies between the maximum release level and the spontaneous release level. IV. Calculation of Results: The following formula is used to calculate "Cytotoxicity Percentage". Cytotoxicity Percentage (%) = (A Sample - A Spontaneous) / (A Maximum Release - A Spontaneous) times 100. A Sample, absorbance of the sample treated with toxic reagent at 565 nm; A Spontaneous, absorbance of spontaneous release of the sample at 565 nm; A Maximum Release, absorbance of maximum release of the sample at 565 nm. |
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| Theory | Lactate dehydrogenase (LDH) is an oxidoreductase that catalyzes the interconversion of pyruvate and lactate, as well as the interconversion of NADH and NAD+. LDH is a stable enzyme present in all cell types and rapidly released into the cell culture medium upon plasma membrane damage. Therefore, LDH is the most widely used marker in cytotoxicity studies. The Cytotoxicity Detection Kit (Lactate Dehydrogenase Method) provides a simple colorimetric assay for studying cytotoxicity. This assay is based on a typical cytotoxicity assay in which target cells are incubated with cytotoxic chemicals or cytotoxic cells (NK cells, cytotoxic T cells) to induce target cell death and LDH release. The supernatant containing LDH is transferred to the wells of a new 96-well assay plate and mixed with the LDH Reaction Solution. In the presence of LDH, NADH reacts with the tetrazolium salt MTT to generate NAD+ and the reduced form of MTT. This reduced form of MTT exhibits a maximum absorption at 565 nm, and the resulting color intensity directly correlates with the number of cells lysed. After incubation at room temperature for 30 min, the absorbance was read at 565 nm using a microplate reader. | |||||||||||||||||||||||||||||
| Composition |
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| Background | Assessment of cell death or cytotoxicity is often based on the quantification of plasma membrane damage. LDH is a stable enzyme present in all cell types and is rapidly released into the cell culture medium following plasma membrane damage. Therefore, LDH is the most widely used marker in cytotoxicity studies. | |||||||||||||||||||||||||||||
| General Notes | 1. Do not mix components from different batches or manufacturers; otherwise, abnormal results may occur. 2. Avoid creating bubbles when mixing or reconstituted components. 3. Change pipette tips frequently to avoid cross-contamination between components. 4. Before beginning the experiment, ensure that all components and equipment are at the appropriate temperature. 5. Maintain good laboratory habits and wear protective equipment such as gloves, lab coat, mask, and goggles before beginning the experiment. |
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| Storage Temp. | -20℃, valid for 6 months. | |||||||||||||||||||||||||||||
| Test Range | 10,000-100,0000 cells/well |
