| Description | Reactive oxygen species (ROS) are natural by-products of normal oxygen metabolism, including superoxide radicals, hydrogen peroxide, and their downstream products such as peroxides and hydroxides. Studies show that over 95% of ROS in organisms originate from mitochondria. An imbalance leading Reactive oxygen species (ROS) are natural by-products of normal oxygen metabolism, including superoxide radicals, hydrogen peroxide, and their downstream products such as peroxides and hydroxides. Studies show that over 95% of ROS in organisms originate from mitochondria. An imbalance leading to oxidative stress is associated with cell growth, proliferation, development, differentiation, aging, apoptosis, and many physiological and pathological processes. Under normal conditions, a balance exists between the intracellular antioxidant defense system and oxygen free radicals, maintaining ROS at low physiological levels. Under pathological conditions, this balance is disrupted, leading to excessive intracellular ROS levels. This can damage mitochondrial enzymes, lipids, and nucleic acids, causing oxidative stress. Additionally, ROS can attack mitochondrial DNA, causing oxidative damage that leads to structural and functional changes such as reduced mitochondrial ATP synthesis and disrupted mitochondrial membrane potential. Mitochondrial Reactive Oxygen Species (ROS) Production Rate Assay Kit (Fluorometric Method) provides a simple, sensitive, and rapid method for detecting mitochondrial ROS production rate. The principle utilizes the fluorescent probe DCFH-DA for ROS detection. DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) diffuses across the mitochondrial membrane and is hydrolyzed by esterases inside the mitochondria to form non-fluorescent DCFH. DCFH is then oxidized by ROS to generate fluorescent DCF. The rate of increase in DCF fluorescence intensity is proportional to the rate of ROS production.M1492773Component96TStorageM1492773AExtraction Buffer60 mL×22-8℃M1492773BReagentⅠ50 mL2-8℃M1492773CReagent Ⅱ1.5 mL-20℃. Store in the dark.M1492773DReagent Ⅲ1EA2-8℃. Store in the dark.M1492773EReagent Ⅳ1EA2-8℃. Store in the dark.M1492773FReagent Ⅴ1EA2-8℃. Store in the dark.M1492773GReagent Ⅵ20 µL-20℃. Store in the dark.Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.User-Provided Instruments and ConsumablesAdjustable pipettes and tipsHomogenizer, Low-temperature centrifuge, 96-well solid black or solid white microplateConstant temperature incubator, Multifunctional microplate readerExperimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction BufferReady-to-use; equilibrate to room temperature before use.Store at 4°CReagentⅠReady-to-use; equilibrate to room temperature before use.Store at 4°CReagentⅡReady-to-useStore at -20°C protected from light.ReagentⅢPrepare before use: Dissolve contents for 96 tests in 6 mL Reagent I. Mix well.Unused dissolved Reagent III can be stored at 4°C protected from light for 1 month.ReagentⅣPrepare before use: Dissolve contents for 96 tests in 6 mL Reagent I. Mix well.Unused dissolved Reagent IV can be stored at 4°C protected from light for 1 month.ReagentⅤPrepare before use: Dissolve contents for 96 tests in 6 mL Reagent I. Mix well.Unused dissolved Reagent V can be stored at 4°C protected from light for 1 month.ReagentⅥReagent VI is somewhat irritating; personal protection is recommended during use.Working ReagentⅥPrepare before use: Dilute Reagent VI 300-fold with Reagent I according to the required volume.Diluted Working Reagent VI cannot be reused.2. Sample Preparation (Tissue/Cell Mitochondria Extraction)2.1 Weigh approximately 0.1 g of tissue or collect 5 million cells. Add 1 mL of Extraction Buffer and 10 µL of Reagent II. Homogenize on ice using a homogenizer. Centrifuge at 600 g, 4°C for 5 minutes. Collect the supernatant into a new centrifuge tube, discard the pellet.2.2 Centrifuge the supernatant again at 11,000 g, 4°C for 10 minutes. The pellet contains the extracted mitochondria.2.3 Discard the supernatant. Resuspend the pellet in 200 µL of Reagent I. Keep on ice for immediate assay.Notes:(1) Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month.(2) Extracted mitochondrial samples must be assayed on the same day and should not be frozen.(3) For protein concentration determination, Aladdin B774074 Bradford Protein Assay Kit or B406195 Bradford Assay Solution (Ready-to-Use) [for Protein Determination] is recommended.3. Assay Steps3.1 Pre-heat the multifunctional microplate reader to 37°C. Set the fluorescence excitation wavelength to 488 nm and emission wavelength to 525 nm.3.2 Add reagents to a 96-well solid black or solid white microplate as follows:ReagentBlank Well (µL)Test Well (µL)Sample020ReagentⅠ200ReagentⅢ5050ReagentⅣ5050ReagentⅤ5050Working ReagentⅥ30303.3 Mix well. Incubate at 37°C protected from light for 15 minutes.3.4 After incubation, measure the fluorescence intensity over 10 minutes using the microplate reader (Ex/Em = 488/525 nm). Maintain the instrument temperature at 37°C. Record the fluorescence change over 10 minutes.Notes:(1) Fluorescence intensity changes must be measured at a constant 37°C over 10 minutes.(2) When mixing with a pipette, pipette gently to avoid generating bubbles.(3) Use solid black or white 96-well plates to prevent interference between adjacent wells. 4. Result Calculation 4.1 Data Processing Perform linear regression analysis on the sampled data points (fluorescence intensity vs. time) to calculate the regression coefficient, i.e., the slope (k) of the line. The actual mitochondrial ROS production rate equals the slope (k test ) from the linear regression of the sample's fluorescence intensity vs. time data points minus the slope (k blank ) from the linear regression of the background fluorescence intensity vs. time data points. k = (RFU 10min - RFU 0min ) / 600 (assuming time in seconds; 10 min = 600 s) 4.2 Activity Calculation Note: We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas. (1) Based on sample mass: (1) Based on sample mass: ROS Production Rate (RFU/s/g fresh weight) = (k test - k blank ) ÷ (V sample ÷ V total × W) = 100 × (k test - k blank ) (2) Based on sample protein concentration: ROS Production Rate (RFU/s/mg prot) = (k test - k blank ) ÷ (V sample ÷ V total × Cpr) = 10 × (k test - k blank ) ÷ Cpr (3) Based on cell count: ROS Production Rate (RFU/s/10⁴ Cells) = (k tes t - k blank ) ÷ (500 × V sample ÷ V total ) = (k test - k blank ) ÷ 50 Parameter Description: V sample : Sample volume added, 0.02 mL V total : Total resuspension volume of the sample, 0.2 mLCpr: Sample protein concentration, mg/mLW: Sample mass, 0.1 g500: Cell count, in units of 10⁴Precautions1.Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.2.This product is for scientific research use only. Not intended for clinical diagnosis... Read More | Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) uses RedNucleus I staining to detect cell cycle. RedNucleus I is a far-infrared nucleic acid dye with cell membrane permeability, which can quickly enter living cells, specifically bind to DNA, and perform cell cycle detection on living cells without RNase digestion. Compared with the traditional PI staining method, the cells do not need to be broken or fixed, and the operation is simpler. RedNucleus I is a fluorescent dye of double-stranded DNA, and the fluorescence intensity after binding to double-stranded DNA is proportional to the content of double-stranded DNA. The intracellular DNA content can be measured by flow cytometry, and then the cell cycle analysis can be carried out according to the distribution of DNA content. After RedNucleus I staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing two copies of genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1-2. In addition, RedNucleus I is compatible with dyes such as Horizon BV / BUV, FITC and R-PE, and can be periodically detected after sample staining.The kit is usually used to detect the cell cycle of cultured adherent or suspended cells. If it is used for cell cycle detection of tissues, the tissues must be digested into a single cell state.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. this product is applicable to the detection of living cells and fixed cell cycle with certain limitations. Whether it is applicable to different types of cells needs to be determined after testing. If fixation is needed, it is recommended to use ice bath pre cooling 75-80% ethanol -20 ℃ to fix cells overnight. 3. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Instruction： Experimental materials ( self-provided )：①cell lines or other cell samples ( self-prepared ) ；②This kit ; ③ trypsin ( self-prepared ) ;④ Cell culture medium containing FBS ( self-prepared ) ; Experimental procedure： 1.Preparation of cell samples : ( 1 ) ( This step is for adherent cells, if suspended cells, can be carried out directly step ( 2 ) ) Digest cells with trypsin, add cell culture medium, gently blow away cells, collected into the centrifuge tube. Note : The number of cells on the machine needs to reach 50,000 and above, so the initial number of cells collected needs to be sufficient. ( 2 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. Carefully remove the supernatant, add about 1 mL of ice bath pre-cooled 1 × staining buffer ( 10 × staining buffer diluted with diH2O at 1 : 10 ), re-suspend the cells. Repeat once. ( 3 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. After the supernatant was discarded, 1 mL of culture medium was added to re-suspend the cells ( for fixed cells, 1 × PBS can also be used to re-suspend ). Gently flick the bottom of the centrifuge tube to properly disperse the cells to avoid cell aggregation. 2.Staining : 4 µL of RedNucleus I staining solution was added to each tube of cell samples, slowly and fully mixed, and incubated at room temperature in dark for 20 min ( or incubated at 37 ° C in dark for 5-10 min ). The optimal incubation time of different cells is different, and the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. 3.Flow cytometry detection and analysis : Excited at 638 nm by flow cytometry, it is recommended to detect in RL3 or FL4 channels, or use RL1 and RL2 channels. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software.Scope of application：Cell cycle detection... Read More | Inquire | DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED Array (470 nm)Photo KitAlysis Reaction BlockTorque screwdriverSmall screwdriver to easily remove torqued screws after reaction is completeFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity0-30 mA variable LED output3 different LED options: blue (470 nm, included), green (527 nm, sold separately), and white (sold separately)Non-magnetic LED baseChemically resistant LED coverPTFE coated cablingDesigned to be used withPhoto KitAlysis High-Throughput Reaction Screening Kit(sold separately).Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More | Product content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligationProduct content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligation of DNA sticky or flush ends in 5 minutes at room temperature (25°C). The kit contains Quick T4 DNA Ligase and 2×Quick Ligation Reaction Buffer optimized for fast and efficient DNA ligation.The ligation efficiency of Quick Ligation is equivalent to 1 hour of conventional ligation with T4 DNA Ligase. The Quick Ligation products can be used directly in routine bacterial transformation experiments.matters needing attention1. This kit enables most of the linkage reactions to reach the reaction endpoint within 5 minutes or less at 25°C. Increasing the reaction time will not enhance the reaction efficiency. If you use the rapid connection reaction after 1 hour, the conversion efficiency will be significantly reduced; if the rapid connection reaction at 25 ℃ overnight, the conversion efficiency will drop to 75%.2. 2×Quick Ligation Reaction Buffer contains ATP, which should be thawed on ice and mixed thoroughly before use. It is recommended to freeze the buffer in small tubes for the first time, so as to avoid repeated freezing and thawing, which will affect the efficiency of DNA ligation.3. Since T4 DNA Ligase contains glycerol, which is sticky and easy to hang on the wall, it is recommended to collect the liquid to the bottom of the tube by centrifugation for a short period of time before use, and the tip of the lance should not go too deep into the liquid surface when taking samples to avoid sticking to the tip of the lance and causing losses.4. If the quick ligation product is used for electrotransformation, the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation, and it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation.Usage1. The reaction solution was prepared according to the following system:*The amount of Insert DNA used: the molar ratio of Vector DNA and Insert DNA is generally 1:3-1:8, and the appropriate molar ratio of Vector DNA and Insert DNA can be selected according to the experimental situation.Calculation of DNA molar number: DNA molar number (nmol)=DNA mass (ng)/( 660daltons x number of inserted DNA bases bp).2. mix gently and centrifuge briefly. react at 25°C for 5 minutes.Note: The reaction time should not exceed 15 minutes, otherwise the connection efficiency will be reduced.3. Do not perform heat inactivation reactions. Centrifuge instantly and collect the solution from the wall to the bottom of the tube.Note: Heat inactivation significantly reduces transformation efficiency due to the presence of PEG in the buffer.4. After the reaction, store the DNA ligation product at 0-4℃, and then carry out transformation experiments; you can also store the DNA ligation product at -20℃.Note: When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.5. Heat shock the ligation product to transform 50 µl of receptor cells or take 1-2 µl of ligation product to electroshock transform 50 µl of receptor cells.Note: 1) When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.(2) If the quick ligation product is used for electrotransformation, it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation because the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation... Read More |