| Description | Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) in living organisms. It is a by-product of active oxygen metabolism, primarily produced by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) in living organisms. It is a by-product of active oxygen metabolism, primarily produced by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but also a hub for the interconversion of reactive oxygen species. On one hand, H₂O₂ can directly or indirectly oxidize biological macromolecules like nucleic acids and proteins within cells, damaging cell membranes and thereby accelerating cellular aging and disintegration. On the other hand, H₂O₂ is also a key regulatory factor in many oxidative stress responses. It can activate factors like NF-κB, and these H₂O₂-related signaling pathways are associated with many diseases such as asthma, inflammatory arthritis, arteriosclerosis, and neurodegenerative diseases. H₂O₂ is also closely related to processes like cell apoptosis and proliferation.Detection Principle: H₂O₂ oxidizes ferrous ions (Fe²⁺) to ferric ions (Fe³⁺). The Fe³⁺ then forms a purple complex with xylenol orange in a specific solution. The absorbance at 580 nm is directly proportional to the H₂O₂ concentration, allowing for the quantification of H₂O₂ levels.Detection Range: 1-100 µMSensitivity: 1 µMApplicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma), urine.H1492752Component96T480TStorageH1492752AReaction Buffer5 mL25 mL-20℃. Store in the dark.H1492752BH₂O₂ Standard (1M)0.1 mL0.1 mL-20℃. Store in the dark.H1492752CAssay Buffer (10×)13 mL65 mL2-8℃Please check the quantity of each component before the experiment.An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 580 nm.Consumables96-well Microplate / Ultrafiltration tubesStandard transparent plate / 10 kDa MWCOReagentsPBS (pH 7.4) / Deionized Water / 30% ZnSO₄ solutionFor washing cells/bacteria / Reagent preparation / Protein removalOthersHomogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsReaction BufferReady-to-use; equilibrate to room temperature before use.Protect from light during the experiment; aliquot and store at -20°C in the dark.H₂O₂ Standard (1M)Ready-to-use; equilibrate to room temperature before use.Protect from light during the experiment; aliquot and store at -20°C in the dark.Assay Buffer (1×)Dilute the 10× Assay Buffer 1:10 with deionized water before use; equilibrate to room temperature.The diluted buffer can be stored at 4°C for at least 2 months. Used for diluting H₂O₂ standard and samples.2. Standard PreparationStandard Curve Setup:First, prepare a 2 mM H₂O₂ Standard: Dilute 2 µL of the 1M H₂O₂ Standard with 998 µL of Assay Buffer (1×).Then, prepare a 100 µM H₂O₂ Standard: Dilute 50 µL of the 2 mM H₂O₂ Standard with 950 µL of Assay Buffer (1×).Using the 100 µM H₂O₂ Standard, prepare further dilutions as shown in the table below.Prepare fresh standard solutions for each experiment.Prepared standards must be used within 4 hours.If the sample is a cell suspension, it is recommended to prepare the H₂O₂ standards using the culture medium.Standard Working Solution100µM Standard (µL)Assay Buffer (1×) (µL)Concentration (µM)1200010021001005034016020420180105101905641962721981Blank020003. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. When ready for the experiment, thaw samples on ice. Note that this may affect sample stability, and results might be lower than expected. The following substances interfere with detection and should be avoided in samples: Ferric salts, iron salts, sucrose, glucose, ascorbic acid, SDS (>0.2%), sodium azide.3.1 Animal Tissues:Wash the tissue with cold PBS to remove as much blood as possible. Blot dry, weigh 0.1 g, and add 1 mL of pre-cooled Assay Buffer (1×). Homogenize the sample on ice. Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.2 Plant Tissues:Weigh approximately 0.1 g of sample, add 1 mL of pre-cooled Assay Buffer (1×), and grind. Disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.3 Cells/Bacteria:Collect 5×10⁶ cells or bacteria. Wash with cold PBS, then add 1 mL of pre-cooled Assay Buffer (1×). Homogenize on ice or disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.4 Plasma, Serum, and Urine (and other biological fluids):Remove proteins and use the supernatant. Protein removal methods:Use a 10 kDa ultrafiltration tube: filter and collect the filtrate.Mix sample : 30% ZnSO₄ solution = 20 : 1, vortex, then centrifuge at 10000 g, room temperature for 5 min, and collect the supernatant.4. Assay Steps4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 580 nm.4.2 Assay System Setup:ReagentStandard Well (µL)Test Well (µL)Standard (various conc.)600Sample060Reaction Buffer40404.3 Mix the reaction system thoroughly and incubate at 37°C for 10 minutes.4.4 Absorbance Measurement: Read the absorbance at 580 nm, recorded as A blank, A standard, and A test. 5. Result CalculationThe following provides both the derived formula and the simplified calculation formula, which are completely equivalent.5.1 Data ProcessingCalculate ΔA standard = A standard - A blank, ΔA test = A test - A blank. 5.2 Standard Curve PlottingPlot the standard curve with standard concentration as the y-axis and ΔA <sub> standard </sub> as the x-axis. Substitute ΔA <sub> test </sub> into the equation to obtain the y value (µM).5.3 Sample H₂O₂ Concentration Calculation(1) Based on sample mass:H₂O₂ Content (nmol/g fresh weight) = y × V sample ÷ (W × V sample ÷ V total ) × n = y ÷ W × n(2) Based on cell or bacterial count:H₂O₂ Content (nmol/10⁴ cells) = y × V sample ÷ (500 × V sample ÷ V total ) × n = y ÷ 500 × n(3) Based on liquid volume:H₂O₂ Content (nmol/mL) = y × V sample ÷ V sample × n = y × nParameter Description:1 µM = 1 nmol/mL;V sample : Volume of sample added;V total : Volume of Assay Buffer (1×) added, 1 mL;n: Sample dilution factor;W: Sample mass, g;500: Cell or bacterial count, in units of 10⁴.6. Result PresentationTypical Standard Curve: y = 207.21x + 1.4921, R² = 0.9988Example-1: 0.1 g of corn tissue was processed and assayed according to the procedure using a 96-well plate.Measured: ΔA test = A test - A blank = 0.278 - 0.048 = 0.230Substituting into the standard curve gives y = 49.15 µM.Calculated based on sample mass:H₂O₂ Content (nmol/g) = y ÷ W × n = 491.5 nmol/g.Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.3. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.5. This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked QuestionsQ: What should I do if the sample ΔA <sub> test </sub> is too high or too low?A: If the sample ΔA test is greater than the ΔA standard of the 100 µM standard, the H₂O₂ content in the sample is too high. Dilute the sample appropriately with Assay Buffer (1×) (multiply by the dilution factor in the calculation). If the sample ΔA test is less than 0.005, increase the sample amount... Read More | The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry.The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry. Compared with other fluorescent substrates or fluorescent inhibitors of Caspase based on ( FLICA ) analysis, aladdin 488 Caspase-3 Substrate does not inhibit the apoptosis process of intact cells while detecting Caspase-3 activity. Substrate is composed of fluorescent DNA dyes coupled with Caspase-3 DEVD recognition sequence. Substrate initially had no fluorescence and entered the cytoplasm through the cell membrane. In apoptotic cells, Caspase-3 cleaves the Substrate and releases high-affinity DNA staining, which migrates to the nucleus to label DNA and emits bright green fluorescence.Therefore, aladdin 488 Caspase-3 Substrate is bifunctional, which can not only detect Caspase-3 activity, but also visualize the morphological changes of the nucleus during apoptosis. Aladdin 488 staining can be fixed in formaldehyde and compatible with subsequent immunostaining experiments.Parameters:aladdin 488：Ex/Em = 500/530 nm (with DNA)Component：Points for attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments. 2.Cells can be co-stained with a final concentration of 1µM Hoechst 33342 dye to produce blue fluorescence staining of the nucleus ( Ex / Em = 346 / 460 nm ). 3.Aladdin 488 staining can be fixed by formaldehyde, but it is not compatible with methanol fixation. 4.Formaldehyde-fixed aladdin 488-stained cells can be treated with 0.1 % TritonX-100 for subsequent staining, but the brightness of the treated staining may be weakened. 5.Fluorescent dyes all have quenching problems, please try to avoid light to slow down the fluorescence quenching. 6.For your safety and health, please wear experimental clothes and wear disposable gloves.Scope of application：Caspase 3 kit and apoptosis detectionUsage:1. Experimental optimization: The experimental steps provided below are based on the endpoint detection system. Aladdin 488 Substrate can also be used for long-term cell incubation course research. Cell density, substrate concentration, and inhibitor concentration may need to be optimized. The optimal substrate concentration may be between 1-10 µ Between M. Cells can be incubated with substrates in culture medium, PBS, or other buffer of your choice. For adherent cells, we recommend replacing them with fresh culture media containing substrates to prevent background heterogeneity. The operation of changing the medium or washing the cells after substrate incubation is freely selectable.2. We suggest that you set the following controls:A. Negative control: cells that do not induce apoptosis;B. Positive control: cells that induce apoptosis;C. Inhibitor control: Induce cell apoptosis while incubating Caspase-3/7 inhibitors (or 10-30 minutes in advance), and finally add Aladdin 488 Caspase-3 substrate.3. The Caspase-3/7 inhibitor Ac-DEVD-CHO in the Ac-DEVD-CHO Caspase-3 inhibitor control kit can be used to confirm that Caspase-3/7 depends on the fluorescence signal of aladdin 488. For inhibitor control, the final concentration of the inhibitor should be at least twice the substrate concentration (e.g. when using 5 µ At substrate M aladdin 488, the concentration of Ac-DEVD-CHO is 10 µ M). Before adding the substrate, incubate Ac-DEVD-CHO at room temperature for 15-30 minutes. After adding the substrate, continue to retain the inhibitor in the incubation solution. Ac-DEVD-CHO is a reversible competitive inhibitor. In certain cell types, effective Caspase-3/7 inhibitors require the use of irreversible inhibitors, such as Z-DEVD-FMK, or the addition of inhibitors before or during apoptosis induction.4. Flow cytometry(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.(2) Adhering cells should be digested with trypsin or other methods before performing the aladdin 488 Caspase-3 experiment.(3) Resuspend cells with culture medium or buffer to achieve a cell density of 106 cells/mL(4) Suck 0.2 mL of cell suspension into a flow cytometry test tube.(5) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(6) 200 µ Add 5 to L cell suspension µ Substrate of 0.2 mM and immediately mix to achieve a substrate concentration of 5 µ M. The optimal substrate concentration for different cells may vary and requires analysis and optimization.(7) Incubate cells at room temperature in dark for 15-30 minutes.(8) Join 300 µ L-medium or PBS, analyzed by flow cytometry. Detect the channel for green fluorescence (Ex/Em=485/515 nm).5. Fluorescence microscope(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.(2) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(3) Using a solution containing 5 µ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.(4) Incubate cells at room temperature for 30 minutes or longer.(5) Cells can be directly observed in culture media containing Substrate. For the endpoint analysis method, PBS was used to clean the cells, fluorescence microscopy was used to observe the cells, and a filter (Ex/Em=485/515 nm) was used to observe green fluorescence.6. Fluorescence enzyme-linked immunosorbent assay (ELISA) reader(1) Adherent cells grow in black 96 well plates; Suspend cells, adjust the density to 106 cells/mL, and divide 0.2 mL of cell suspension into one well.(2) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls. Note: Cells may be processed in tubes or bottles and then transferred to a 96 well detection plate.(3) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(4) For suspended cells, directly add Substrate and mix well. For adherent cells, use a solution containing 5 µ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.(5) Cells can be directly observed in culture media containing Substrate.(6) For suspended cells, gently shake to resuspend the cells. The fluorescence enzyme-linked immunosorbent assay instrument is set with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Suggest using bottom collection method for adherent cells. Changes in the density of adherent cells may lead to inaccurate readings... Read More | Inquire | Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Buffer20 µL-20℃.Avoid freeze/thaw cycle. C666001EDigestion Enzyme I70 µL-20℃.Avoid freeze/thaw cycle. C666001FDigestion Enzyme III25 µL-20℃.Avoid freeze/thaw cycle. Box 2: Magnetic Beads for DNA Purification and RecoveryC666001Component16 TStorageC666001GCMPure4×1.5 mL2-8℃Products IntroductionThe Cyclization Kit is a modular kit tailored for the MGI high-throughput sequencing platform. With this kit, PCR products after junction ligation can be prepared into single-stranded circular DNA libraries suitable for MGI sequencers. All reagents provided in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 (Cat. No. 12321D) is recommended.2. "Qubit" 3.0 Fluorescence Quantimeter (ThermoFisher, Cat. No. Q33216)3. Qubit" ssDNA Assay Kit (Invitrogen, Cat. No. Q10212)4. Anhydrous ethanol, EB (10 mM Tris-HCl, pH 8.0), NF Water (pH between 7.0 and 8.0).5. reaction tubes: low adsorption PCR tubes with 1.5 mIEP tubes are recommended: 5.Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and libraries. Pre-experiment Preparation and Important Notes 1. Sample preparation.PCR product: 2330 ng total (total amount when multiple PCR products are mixed) in a volume of 49 pL (if the volume of PCR product is insufficient, add NF Water to bring the total volume to 49 pl). -PCR product: Fragment size: The fragment peak is between 200-500 bp. -PCR product fragment size: Fragment peaks between 200-500 bp. -PCR product modification: Fixed sequences (with Index) for MGISEQ-2000, MGISEQ-200 and BGISEQ-500 sequencing platforms were added.2. Reagent preparation-Remove the corresponding reagents from the kit, centrifuge briefly, and place the enzyme mixture on ice until ready to use: buffers need to be dissolved at room temperature before use, then centrifuged with shaking and placed on ice until ready to use, and NF Water and EB are placed at room temperature until ready to use: "Please make up the mixture on ice:Precipitation may appear after the buffer in the kit is dissolved, the precipitation does not affect the function of the reagent, please shake and mix well until the precipitation disappears and then use. Schematic diagram of the cyclization process procedurecyclize 1. 1 wl of Splint Oligo was added to the 49JI PCR product. The product was denatured and incubated on a PCR instrument at 95°C for 3 min, then immediately transferred to an ice bath and allowed to stand for 2 min. 2. The reaction mixture was prepared on ice according to the following system. 3. Add 15ul of the above reaction mixture to 50µl of denatured DNA.4. Place the above PCR tubes on the PCR instrument under the following conditions Reaction. digest 1. Prepare the digestion reaction solution on ice according to the following system. 2. After the cyclization reaction, add 8l of digestion reaction solution directly to the cyclization system, mix well, centrifuge briefly and then place the PCR tube on the PCR instrument and react under the following conditions. 3. Purification was carried out immediately after the reaction.Purification of digestive products1. Remove CMPure at room temperature 30 minutes prior to use and mix well with shaking.2. Transfer the digested product to a 1.5 mIEP tube, pipette 340 pICMPure into the digested product, mix well by gently blowing 10 times with a pipette and incubate for 10 minutes at room temperature.3. Instantaneous centrifugation, place the EP tube on a magnetic rack and let stand for 5 minutes until the liquid is clear, pipette and discard the supernatant.4. Keep the EP tube fixed on a magnetic rack, add 250ul of freshly prepared 80% ethanol, let it stand at room temperature for 1 minute, then carefully discard the supernatant.5. Repeat step 4 once, try to suck up the liquid at the bottom of the tube: Note: Do not suck up the magnetic beads, so as not to affect the yield.6. Keep the EP tube fixed on the magnetic rack, open the cap and dry it at room temperature for 5-10 minutes.7. Remove the EP tube from the magnetic rack, add 35ul of EB or NF Water for DNA elution, pipette blow to mix and dissolve at room temperature for 10 min.8. Centrifuge instantaneously, place the EP tube on a magnetic rack and let stand for 2 minutes until the liquid is clarified, transfer the supernatant to a new EP tube. -Store at 20C and leave to prepare DNB... Read More | Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to bind to the silica gel centrifugal adsorbent columns in a highly efficient manner, and the viral nucleic acids obtained are of high purity and stable quality, free of protein, nuclease and other impurities, and can be used in a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments. It can be used for a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments.Bring your own instrumentsThermostatic mixer.Pre-experiment Preparation and Important Notes1. Read these instructions carefully before experimenting.2. If Proteinase K is to be stored for a long period of time, please keep it at -20℃.3. Check Buffer RLC for crystallization or precipitation prior to use, and if crystallization or precipitation occurs, redissolve Buffer RLC in a 56°C water bath.4. Pre-treatment of tissue samples: Take 20 mg of tissue samples into 1.5 mL centrifuge tubes (self-provided), add 500 µL of Buffer RLC, and after the tissue homogenizer breaks up, centrifuge the samples for 1 minute at 12,000 rpm (~13,400×g), and take 200 µL of supernatant as samples. procedure1. Take a 1.5 mL centrifuge tube (provided), add 500 µL of Buffer RLC, 200 µL of sample, 20 µL of Proteinase K, vortex for 5 s, and then place it in a thermostatic mixer at 1200 rpm for 10 min at room temperature. Note: For wet swab samples, 200 µL of sample was taken after sufficiently shaking and mixing. Note: For wet swabs, 200 µL was taken from the sample after it was soaked in 400 µL of saline, shaken and mixed thoroughly for 5 minutes, and then centrifuged at 12,000 rpm for 1 minute, and 200 µL was taken for extraction.2. Instantly remove the centrifuge tube and add the solution from step 1 to the Spin Columns DM in the collection tube. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.3. Add 500 µL of Buffer PGWT to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.4. Add 500 µL of Buffer GWT2 to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.5. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the adsorption column at room temperature for 2 minutes and allow to dry.6. Place the column in a new collection tube (RNase-Free Centrifuge Tube), add 40-100 µL of RNase-Free Water to the center of the column membrane, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect the nucleic acid solution. Store at -80℃ for a long time... Read More |