| 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 | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... Read More | D-Lactate, typically present in the bloodstream at nanomolar concentrations, is produced by an intestinal source or via the methylglyoxal pathway. In mammals, D-Lactate metabolism requires D-Lactate hydrogenase and is metabolized slowly, thus an increase in blood concentration levels can lead to D-Lactate, typically present in the bloodstream at nanomolar concentrations, is produced by an intestinal source or via the methylglyoxal pathway. In mammals, D-Lactate metabolism requires D-Lactate hydrogenase and is metabolized slowly, thus an increase in blood concentration levels can lead to acidemia and acidosis. The severity of this D-lactic acidosis can be associated with neurotoxic symptoms. Significant D-Lactate accumulations in the body can also be related to impaired metabolism and excretion.D-Lactate Colorimetric Assay kit has been used to determine the stereospecificity of lactate produced.Suitability: Suitable for use with samples of serum, plasma, cells, culture and fermentation media.Principle: In this assay, D-Lactate is specifically oxidized by D-Lactate hydrogenase and generates a proportional colorimetric product measured at 450 nm. The useful concentration range in samples is 0.1-10 mM D-Lactate... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Inquire |