• MTS Cell Proliferation And Cytotoxicity Detection Kit
  • MTS Cell Proliferation And Cytotoxicity Detection Kit

LDH Cytotoxicity Assay Kit

This product is an INT color reaction based on diphorase catalysis, which uses colorimetric methods to detect the activity of lactate dehydrogenase released during cytotoxicity or to detect lactate dehydrogenase activity in other samples.
This kit can be used for routine detection of lactate dehydrogenase activity, and is more commonly used for cytotoxicity testing based on LDH release. Meanwhile, based on the detection of total lactate dehydrogenase activity in cells, this kit can also be used for detecting cell proliferation and cytotoxicity.
  • MTS Cell Proliferation And Cytotoxicity Detection Kit

SPECIFICATION

This product is an INT color reaction based on diphorase catalysis, which uses colorimetric methods to detect the activity of lactate dehydrogenase released during cytotoxicity or to detect lactate dehydrogenase activity in other samples.
This kit can be used for routine detection of lactate dehydrogenase activity, and is more commonly used for cytotoxicity testing based on LDH release. Meanwhile, based on the detection of total lactate dehydrogenase activity in cells, this kit can also be used for detecting cell proliferation and cytotoxicity.

Background
The destruction of cell membrane structure caused by cell apoptosis or necrosis can lead to the release of enzymes from the cytoplasm into the culture medium, including lactate dehydrogenase (LDH) with relatively stable enzyme activity. Quantitative analysis of cell toxicity can be achieved by detecting the activity of LDH released from cells with ruptured plasma membranes into the culture medium. LDH release is considered an important indicator of cell membrane integrity and is widely used for cytotoxicity testing. LDH release is considered a safe and effective alternative method for detecting cell membrane integrity through 51Cr release, which was previously used to label cells with radioactive 51Cr.

Detection principle

Under the action of lactate dehydrogenase, NAD+is reduced to produce NADH, and NADH and INT are catalyzed by thiooctane dehydrogenase to produce NAD+and the strong chromophore formazan. An absorption peak is generated at a wavelength of 490nm, allowing for the quantification of lactate dehydrogenase activity through colorimetry. There is a linear positive correlation between absorbance and lactate dehydrogenase activity. The schematic diagram of the enzyme-linked reaction principle is as follows:
Component 100 times /500 times
LDH release reagent 1.5mL 7.5mL
Lactic acid solution 2mL 10mL
Enzyme solution 2 × 1mL 2 × 5mL
INT solution (10X) 200 μ L 1mL
INT diluent 2mL 10mL

Storage: -20 ℃, valid for one year. Enzyme solution should be careful to avoid repeated freezing and thawing. INT solution (10X) needs to be stored away from light. After thawing, the reagent kit can be stored at 4 ℃ for a short period of time and is effective for 2-3 days.
Freezing will inactivate some of the lactate dehydrogenase in the sample, which can be stored at 4 ℃ for 2-3 days. It is recommended to complete the measurement on the same day as much as possible after the sample is prepared.
If detecting lactate dehydrogenase in cell culture medium, it is recommended that the concentration of serum should not exceed 1% due to the presence of lactate dehydrogenase in serum, and it is best to use heat inactivated serum. If it is necessary to use 10% serum, a control well without cells but with the same volume of culture medium must be set during testing to eliminate background.
Excessive cell growth, high density, high centrifugation speed, and large temperature difference inside and outside the incubator can all cause an increase in the release of lactate dehydrogenase by cells.
If you want to conduct absolute quantification of lactate dehydrogenase activity, users need to provide their own lactate dehydrogenase standard.

Sample preparation:

Method 1: LDH release detection
Inoculate an appropriate amount of cells into a 96 well cell culture plate based on their size and growth rate, so that the cell density does not exceed 80-90% when tested.
Suck off the culture medium and wash once with PBS solution. Fresh culture medium (recommended to use low serum culture medium containing 1% serum or appropriate serum-free culture medium), and divide each culture well into the following groups: cell-free culture medium well (background blank control well), control cell well (sample control well) without drug treatment, cell well (maximum enzyme activity control well) for subsequent lysis without drug treatment, and cell well (drug treated sample well) with drug treatment, And mark it properly. Provide appropriate medication treatment according to experimental needs (such as adding 0-10) μ For specific drug stimuli around L, different concentrations and treatment times can be set, and appropriate drug solvents need to be added to the control well for control. Continue to cultivate as usual. One hour before the scheduled detection time, remove the cell culture plate from the cell culture box and add the LDH release reagent provided by the reagent kit to the "maximum enzyme activity control well" of the sample, with a dosage of 10% of the original culture medium volume. After adding the LDH release reagent, repeatedly blow and mix several times, and then continue to incubate in the cell incubator.
After reaching the predetermined time, centrifuge the cell culture plate with 400g in a porous plate centrifuge for 5 minutes. Take 120 pieces of supernatant from each well separately μ L. Add it to the corresponding hole of a new 96 well plate and proceed with sample determination immediately.
Method 2: Detection of total intracellular LDH

Cytotoxicity test: Inoculate an appropriate amount of cells into a 96 well cell culture well plate based on their size and growth rate, so that the cell density does not exceed 80-90% when tested. Add different drugs for treatment and set appropriate controls. After drug stimulation, centrifuge the cell culture plate with 400g in a porous plate centrifuge for 5 minutes. Try to remove the supernatant and add 150 μ Dilute the LDH release reagent provided by the kit with PBS 10 times (add 1 volume of LDH release reagent to 10 volumes of PBS and mix well), shake the culture plate appropriately and mix well, then continue to incubate in the cell incubator for 1 hour. Subsequently, centrifuge the cell culture plate with 400g in a porous plate centrifuge for 5 minutes. Take 120 pieces of supernatant from each well separately μ L. Add it to the corresponding hole of a new 96 well plate and proceed with sample determination immediately.
Cell proliferation detection: According to the size and growth rate of cells, an appropriate amount of cells should be inoculated into a 96 well cell culture well plate, so that the cells are not more than 80-90% full after being stimulated by drugs that promote cell proliferation. Use different drugs to stimulate cells and set appropriate controls. After drug stimulation, centrifuge the cell culture plate with 400g in a porous plate centrifuge for 5 minutes. Try to remove the supernatant and add 150 μ Dilute the LDH release reagent provided by the kit with PBS 10 times (add 1 volume of LDH release reagent to 10 volumes of PBS and mix well), shake and mix the cells appropriately, and then continue to incubate in a fine incubator for 1 hour. Subsequently, centrifuge the cell culture plate with 400g in a porous plate centrifuge for 5 minutes. Take 120 pieces of supernatant from each well separately μ L. Add it to the corresponding hole of a new 96 well plate and proceed with sample determination immediately.
Note: LDH release detection is more commonly used, and total intracellular LDH detection can usually be replaced by methods such as MTT, WST-1, or CCK-8.

Preparation of the reagent kit:
Preparation of INT solution (1X): According to the required amount of INT solution (1X), take an appropriate amount of INT solution (10X) and dilute it with INT diluent to 1X. For example, take 20 μ L INT solution (10X), add 180 μ L INT diluent, mix well and prepare to 200 μ L INT solution (1X). INT solution (1X) should be prepared and used immediately. After preparation, it can be stored at 4 ℃ for use on the same day, and should not be frozen after preparation.
Preparation of LDH testing working solution: According to the number of samples to be tested (including control), refer to the table below to freshly prepare an appropriate amount of testing working solution before testing.
Attention: The LDH detection working solution must be prepared and used immediately, and attention should be paid to avoiding light during preparation and use.
(Optional) If you want to conduct absolute quantification of LDH enzyme activity, you need to prepare your own LDH standard and freshly prepare different concentrations of LDH standard, such as 10mU/ml, 5mU/ml, 2.5mU/ml, 1.25mU/ml, 0.65mU/ml, and 0 mU/ml.
Sample determination:
Add 60 to each hole separately μ L LDH detection working solution.
Mix well and incubate at room temperature (about 25 ℃) in dark for 30 minutes (can be wrapped in aluminum foil and placed on a horizontal or side sway shaker to slowly shake). Then measure the absorbance at 490nm. Use any wavelength of 600nm or greater as the reference wavelength for dual wavelength measurement.
Calculation (the absorbance of each group measured should be subtracted from the absorbance of the background blank control hole)
Cytotoxicity or mortality rate (%)=(absorbance of treated sample - absorbance of sample control well)/(absorbance of maximum enzyme activity of cells - absorbance of sample control well) × one hundred
Can draw a cytotoxicity curve: the vertical axis represents the actual absorbance, and the horizontal axis represents the drug concentration; Based on this, the half lethal dose LD50 of the drug at a specific time of action can be calculated.
Appendix 2:
If it is necessary to accurately calculate the absolute activity of LDH enzyme, a standard curve can be drawn using a series of LDH standards and corresponding measured absorbance values. The LDH enzyme activity in the sample can be calculated using the corresponding formula of the standard curve.
After subtracting the blank control from the values of each well, draw the LDH standard curve with the detected absorbance (OD490) as the vertical axis and LDH enzyme activity (mU) as the horizontal axis. Simultaneously calculate the formula for the trend line.
Cytotoxicity test kit (lactate dehydrogenase method)
A490nm=k × LDH enzyme activity unit (mU)+b, calculate the slope k and intercept b of the trend line using software such as Excel.
Calculate the LDH activity in the sample according to the above formula.
Actual absorbance of the sample (OD490)=absorbance measured by the sample well - absorbance of the background blank control well
LDH enzyme activity unit (mU) in the detection system=(OD490-b)/k
LDH enzyme activity in the sample (mU/ml)=LDH enzyme activity unit in the detection system (mU)/detection sample volume
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