Lactate dehydrogenase cytotoxicity test kit Product number product name package C0017 Lactate dehydrogenase cytotoxicity test kit 500 times Product introduction: A LDH Cytotoxicity Assay Kit, also known as the LDH Assay Kit or the LDH Release Assay Kit, is A kit based on diaphorase-catalyzed INT color reaction, which detects lactic acid dehydrogenase activity released by cytotoxicity or detects lactate dehydrogenase activity in other samples by colorimetry. (lactate dehydrogenase, LDH). Quantitative analysis of cytotoxicity can be achieved by detecting the activity of LDH released into the culture fluid from cells disrupted by the plasma membrane. LDH release is seen as an important indicator of cell membrane integrity and is widely used for cytotoxicity testing. LDH release is considered to be a safe and effective alternative to the use of radioactive 51Cr-labeled cells, followed by 51Cr release for cell membrane integrity testing. The basic principle of this kit is that under the action of lactate dehydrogenase, NAD + is reduced to produce NADH, NADH and INT (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride) are diaphorase The catalytic reaction produces NAD + and the strong coloring matter formazan, which produces an absorption peak at a wavelength of 490 nm, so that the activity of lactate dehydrogenase can be quantified by colorimetry. The absorbance is linearly positively correlated with lactate dehydrogenase activity. The schematic diagram of the principle of the enzyme-linked reaction is as follows: Ø It can detect the activity of lactate dehydrogenase in samples such as cell culture fluid and cell lysate. A kit can be tested 500 times. packing list: Product number product name package C0017-1 LDH release reagent 7.5ml C0017-2 Lactic acid solution 10ml C0017-3 Enzyme solution 5ml×2 C0017-4 INT solution (10X) 1ml C0017-5 INT diluent 10ml - Instruction manual 1 copy Storage Conditions: Save at -20oC, valid for one year. C0017-3 should take care to avoid repeated freezing and thawing. C0017-4 needs to be stored away from light. The kit can be stored for a short period of 4oC after thawing and is valid for 2-3 days. Precautions: Freezing will inactivate some of the lactate dehydrogenase in the sample and can be placed for 2-3 days at 4oC. It is recommended that the sample be prepared as soon as possible after the sample is prepared. If the lactate dehydrogenase in the cell culture medium is detected, since the serum contains lactate dehydrogenase, it is recommended that the serum concentration should not exceed 1%, and it is preferable to use heat inactivated serum. If 10% serum is necessary, it is necessary to set up no cells, but add control wells of the same volume to remove the background. If you want to perform absolute quantification of lactate dehydrogenase activity, you need to bring your own lactate dehydrogenase standard. For your safety and health, wear a lab coat and disposable gloves. Instructions for use: Sample preparation: Method 1: LDH release detection a. Inoculate a suitable amount of cells into a 96-well cell culture plate according to the size and growth rate of the cells so that the cell density is not more than 80-90% when tested. The culture solution was aspirated and washed once with PBS. Change the fresh medium (recommended to use low serum culture medium containing 1% serum or appropriate serum-free medium), and divide each culture well into the following groups: including cell-free culture wells (background blank control wells), Drug-treated control cell wells (sample control wells), drug-treated cell wells for subsequent lysis (sample maximum enzyme activity control wells), and drug-treated cell wells (drug-treated sample wells) are labeled . According to the needs of the experiment, appropriate drug treatment should be given (such as adding 0-10μl of specific drug stimulation, different concentrations can be set, different treatment time, appropriate drug solvent control should be added to the control well), and continue to be cultured. One hour before the scheduled detection time point, the cell culture plate was taken out from the cell culture incubator, and the LDH release reagent provided by the kit was added to the "sample maximum enzyme activity control well" in an amount of 10% of the volume of the original culture solution. . After the LDH release reagent is added, the mixture is repeatedly blown several times and then continued to incubate in the cell culture incubator. After the predetermined time was reached, the cell culture plates were centrifuged for 5 min in a multiwell plate centrifuge at 400 g. Take 120 μl of the supernatant from each well and add to the corresponding well of a new 96-well plate. In the middle, the sample is measured immediately. Method 2: Detection of total LDH in cells Cytotoxicity assay: Appropriate cells are seeded into 96-well cell culture wells according to cell size and growth rate so that the cell density is not more than 80-90% when tested. Add different drugs for treatment and set appropriate controls. After the drug stimulation was completed, the cell culture plates were centrifuged for 5 min in a multiwell plate centrifuge at 400 g. Try to aspirate the supernatant, add 150 μl of the LDH release reagent provided by the kit diluted 10 times with PBS (add 1 volume of LDH release reagent to 10 volumes of PBS and mix), shake the plate as appropriate, and continue in the cell culture. Incubate for 1 hour in the box. The cell culture plates were then centrifuged for 5 min using a multiwell plate centrifuge at 400 g. 120 μl of the supernatant of each well was taken and added to the corresponding well of a new 96-well plate, and the sample was measured. Cell proliferation assay: appropriate cells are seeded into a 96-well cell culture plate according to the size and growth rate of the cells, so that the cells that promote cell proliferation are stimulated to be no more than 80-90%. Use different drugs to stimulate the cells and set up appropriate controls. After the drug stimulation was completed, the cell culture plates were centrifuged for 5 min in a multiwell plate centrifuge at 400 g. Try to aspirate the supernatant, add 150 μl of the LDH release reagent provided by the kit diluted 10 times with PBS (add 1 volume of LDH release reagent to 10 volumes of PBS and mix), mix well by shaking, and continue in the cell culture incubator. Incubate for 1 hour. The cell culture plates were then centrifuged for 5 min using a multiwell plate centrifuge at 400 g. 120 μl of each well supernatant was taken and added to the corresponding well of a new 96-well plate, and the sample was measured. Note: LDH release detection is more common. Intracellular total LDH detection can usually be replaced by MTT, WST-1 or CCK-8. 2. Preparation of the kit: a. Configuration of INT solution (1X): Depending on the amount of INT solution (1X) required, an appropriate amount of INT solution (10X) was diluted to IX with INT dilution. For example, take 20μl INT To the solution (10X), add 180 μl of INT dilution and mix to prepare 200 μl of INT solution (1X). INT solution (1X) should be used now, and it can be stored at 4oC after configuration. Used in days, it is not suitable to be frozen after storage. b. Preparation of LDH test working fluid: According to the number of samples to be measured (including control), refer to the following table to prepare an appropriate amount of test working fluid before the test. Note: LDH The test working fluid must be ready for use, and care should be taken to avoid proper light during preparation and use. Number of detections 1 time 10 times 20 times 50 times Lactic acid solution 20μl 200μl 400μl 1ml INT solution (1X) 20μl 200μl 400μl 1ml Enzyme solution 20μl 200μl 400μl 1ml total capacity 60μl 600μl 1.2 ml 3ml c. (Optional) If you want to perform absolute quantification of LDH enzyme activity, you need to prepare LDH standard and freshly prepare different concentrations of LDH standards, such as 10mU/ml, 5mU/ml, 2.5 mU/ml, 1.25 mU/ml, 0.65 mU/ml, 0 mU/ml. 3. Sample determination : a. 60 μl of LDH test solution was added to each well. b. Mix and incubate at room temperature (about 25oC) for 30 minutes in the dark (can be wrapped in aluminum foil and shaken slowly on a horizontal shaker or side swing shaker). The absorbance was then measured at 490 nm. Dual wavelength measurement was performed using any wavelength of 600 nm or more as a reference wavelength. c. Calculation (measured absorbance of each group should be subtracted from background blank control well absorbance) d. Cytotoxicity or mortality (%) = (treatment sample absorbance - sample control well absorbance) / (absorbance of cell maximum enzyme activity - sample control well absorbance) × 100 e. The cytotoxicity curve can be drawn: the ordinate is the actual absorbance, and the abscissa is the drug concentration; according to this, the LD50 of the semi-lethal dose of the drug at a specific time can be calculated. Appendix 1 : The absorbance value corresponding to a known concentration of LDH enzyme standard can be simultaneously determined. The LDH enzyme activity in the sample is roughly calculated by the following formula: LDH activity unit (mU/ml) in the sample to be tested = (sample hole absorbance - background blank control) Hole absorbance) / (Standard tube absorbance - standard blank tube absorbance) × standard concentration (mU / ml); according to the calculation results can be compared between different sample treatment groups for statistical differences. Appendix 2 : To accurately calculate the absolute activity of LDH enzyme activity, a standard curve can be drawn from a series of LDH standards and corresponding measured absorbance values, and the enzyme activity of LDH in the sample can be calculated by the corresponding formula of the standard curve. After subtracting the blank control from each well value, the LDH standard curve was drawn by taking the absorbance (OD490) as the ordinate and the LDH enzyme activity (mU) as the abscissa. The formula for the trend line is also calculated. A 490nm = k × LDH enzyme activity unit (mU) + b, the slope k of the trend line and the intercept b are calculated by software such as Excel. The LDH activity in the sample was calculated according to the above formula. Actual absorbance of the sample (OD490) = absorbance measured in the sample well - background blank control well absorbance LDH enzyme activity unit (mU) = (OD490-b) / k in the detection system LDH enzyme activity (mU/ml) in the sample = LDH enzyme activity unit (mU) / test sample volume in the detection system Frozen Squid Flower Cut,Frozen Squid Flower,Squid Carving Flower,Pineapple Cut Squid Flower Zhoushan Haiwang Seafood Co., Ltd. , https://www.haiwangseafoods.com