| Description | IntroductionHexokinase (HK, EC 2.7.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which serves as the intersection point of glycolysis and the IntroductionHexokinase (HK, EC 2.7.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which serves as the intersection point of glycolysis and the pentose phosphate pathway.Assay PrincipleHK catalyzes the synthesis of Glucose-6-Phosphate (G6P) from Glucose. Glucose-6-Phosphate Dehydrogenase (G6PDH) then further catalyzes the dehydrogenation of G6P, generating NADPH. NADPH has a characteristic absorption peak at 340 nm.Component48T96TStorageExtraction Buffer60 mL60 mL×22-8℃Reagent Ⅰ14 mL28 mL2-8℃Reagent Ⅱ1EA1EA-20℃. Store in the dark.Reagent Ⅲ1EA1EA-20℃. Store in the dark.Note: Please check the quantity of all components before starting the experiment. An additional 10% of each component is provided for standard curve preparation or pilot experiments.Required Materials and Equipment (Not Provided)TypeNameNotesInstrumentMicroplate ReaderMust be capable of measuring absorbance at 340 nmConsumables96-well UV PlateUV-transparent plateReagentsPhysiological SalineFor sample washingOtherHomogenizer (for tissue samples), Incubator, Ice box, Refrigerated centrifuge, Adjustable pipettes and tipsUsing a multichannel pipette is recommended for high-throughput experiments to improve efficiencyInstructions for Use1. Reagent Preparation试剂名称 Reagent NamePreparationNotesExtraction BufferReady-to-use; equilibrate to room temperature (RT) before use.Store at 4°CReagent ⅠReady-to-use; equilibrate to room temperature (RT) before use.Store at 4°CWorking Reagent ⅡPrepare immediately before use:• For 48T: Dissolve contents of Reagent Ⅱ in 10.8 mL Reagent Ⅰ• For 96T: Dissolve contents of Reagent Ⅱ in 21.6 mL Reagent ⅠKeep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles.Working Reagent ⅢPrepare immediately before use:1. Dissolve Reagent Ⅲ: • For 48T: in 0.5 mL Reagent Ⅰ • For 96T: in 1 mL Reagent Ⅰ2. Dilute the dissolved Reagent Ⅲ 10-fold with Reagent Ⅰ to make the Working Reagent Ⅲ.Keep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles.2. Sample PreparationNote: The use of fresh samples is highly recommended. HK activity decreases significantly upon sample freezing.2.1 Animal/Plant TissuesWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.2 Cells, Bacteria, or FungiCollect 5×10⁶ cells/bacteria/fungi. Wash with pre-cooled physiological saline and centrifuge at 800 g for 2 min. Discard the supernatant. Add 1 mL of Extraction Buffer and disrupt the cells by sonication on ice (5 min total, 20% power or 200 W, pulse 3s on/7s off, repeat 30 times). Centrifuge the lysate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.3 Serum (or Plasma)Assay directly.3. Assay Procedure3.1 Microplate Reader Preparation: Preheat for at least 30 min. Set the wavelength to 340 nm.3.2 Working Solution Preparation: Prepare immediately before use. Each well requires 190 µL of Working Solution. It is recommended to prepare enough for 2 extra wells to account for pipetting loss.For a single well: Mix 180 µL of Working Reagent Ⅱ with 10 µL of Working Reagent Ⅲ.The Working Solution must be prepared fresh. Incubate it at 37°C for 10 min before the assay.3.3 Assay Setup: Pipette into wells of the 96-well UV plate as follows:ReagentTest Well (µL)Sample10Working Solution1903.4 Absorbance Measurement: Immediately after adding the Working Solution, mix thoroughly and measure the absorbance at 340 nm at 10 seconds (A₁) and again after exactly 10 minutes of incubation at 37°C (at 10 min 10 sec, A₂).4. Result CalculationThe following derived and simplified calculation formulas are provided and are equivalent.4.1 Data ProcessingCalculate ΔA = A₂ - A₁.4.2 Sample HK Activity Calculation① Based on Sample Mass (U/g)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of sample.Calculation:HK (U/g) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × W) ÷ T × nSimplified Formula: HK (U/g) = 643.09 × ΔA ÷ W × n② Based on Cell/Bacteria/Fungi Count (U/10⁴)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells/bacteria/fungi.Calculation:HK (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × 500) ÷ T × nSimplified Formula: HK (U/10⁴) = 643.09 × ΔA ÷ 500 × n③ Based on Liquid Volume (U/mL)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milliliter of liquid.Calculation:HK (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T × nSimplified Formula: HK (U/mL) = 643.09 × ΔA × n④ Based on Protein Concentration (U/mg prot)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milligram of protein.Calculation:HK (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T × nSimplified Formula: HK (U/mg prot) = 643.09 × ΔA ÷ Cpr × nParameter Description:ε: NADPH molar extinction coefficient = 6.22 × 10³ L/mol/cmd: Light path of the 96-well UV plate = 0.5 cm10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol)Vₜₒₜₐₗ: Total reaction volume = 200 µL = 2 × 10⁻⁴ LVₛₐₘₚₗₑ: Volume of supernatant added to the reaction = 10 µL = 1 × 10⁻⁵ LVₛₐₘₚₗₑₜₒₜₐₗ: Volume of Extraction Buffer added = 1 mLCpr: Sample protein concentration (mg/mL)W: Sample mass (g)T: Reaction time = 10 min500: Cell/Bacteria/Fungi count = 5 × 10⁶, expressed in units of 10⁴n: Sample dilution factorPrecautionsBefore formal testing, it is recommended to perform a pilot experiment using 2-3 samples expected to have significant activity differences.For tissue and cell samples, protein concentration measurement can be used to normalize results between samples.This kit is compatible with spectrophotometer detection. Adjust the preparation volumes of detection reagents proportionally according to the spectrophotometer's requirements.Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear a lab coat, mask, gloves, head cover, and other protective equipment. Perform experiments in a fume hood or biosafety cabinet.This product is for scientific research use only. It is not intended for clinical diagnosis.Frequently Asked Questions (FAQ)1. What should I do if the measured ΔA is too high or too low?If ΔA > 0.5, the sample HK activity is too high. Dilute the supernatant appropriately with Extraction Buffer (include the dilution factor *n* in the calculation) or shorten the reaction time to 5 min.If ΔA < 0.005, the sample HK activity is too low. Increase the sample volume (keep the Working Solution volume constant and adjust the variable in the formula accordingly) or extend the reaction time (e.g., to 15 or 30 min).2. Can multiple samples be assayed simultaneously for high-throughput detection?The initial reaction rate is fast. It is not recommended to run a large number of samples simultaneously. If a multichannel pipette is unavailable, it is best for two individuals to perform the experiment together—one person timing and the other measuring the absorbance—to ensure the accuracy of the results... Read More | Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-ethynyl-2 '- deoxyuridine) is a pyrimidine analog that integrates into the DNA duplex during DNA synthesis. Edu detection is based on the "click" reaction. A copper catalyzed azide reacts covalently with alkynes to form covalent bonds. In this kit, edu contains alkynes, Aladdin ® 488 / 555/594/647a azide dyes contain azide compounds. The edu labeling proliferation of click method is rapid and effective, and easy to use. BrdU method requires DNA denaturation (such as acid denaturation, thermal denaturation or digestion with DNase) to expose BrdU, so as to facilitate BrdU antibody binding; The edu method only needs paraformaldehyde fixation and Triton X-100 penetration to make the detection reagent enter the cells, and only a small amount of azide dye is needed to label the integrated edu very effectively. This kit contains all components required for edu method detection, and can be used for proliferation detection of cultured cells in vitro.Component:Product parameters：555/565 nm；Instruction： Experimental materials (self provided). 10 mM PBS, pH 7.2-7.6. 4% paraformaldehyde fixing solution (in PBS)Propensive reagent (0.5% Triton X -100 in PBS). 2 mg/mL glycine solution (in ddH2O). 3% BSA in PBS, pH 7.2-7.6. 1% BSA in PBS, pH 7.2-7.6. ddH2O. 96/24/12/6 well culture plate or dishFluorescence microscopy detection method1. Cell cultureTake logarithmic growth stage cells and inoculate them into a 96 well plate with 4 × 103-1 × 105 cells per well (the number and density of cells can be adjusted according to cell size, growth rate, and specific requirements of experimental treatment), and culture until normal growth stage.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU marking(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations, such as 10-50, should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash the cells twice with a 3% BSA washing solution after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, remove the culture medium. Wash cells twice with 1X PBS for 5 minutes each time to remove EdU residues that have not been incorporated into DNA. Cells with weak adhesion can reduce cleaning intensity. Join 50 µ After incubating at room temperature for 20 minutes with 4% paraformaldehyde fixative, remove the fixative.(2) Add 50 to each hole µ L 2 mg/mL glycine solution, incubate at room temperature for 5 minutes, and neutralize the remaining fixed solution.(3) At a rate of 100 per hole µ Wash cells twice with 3% BSA.(4) Remove the washing solution and add 100 µ L 0.5% Triton X -100, incubate at room temperature for 10 minutes.5. EdU detectionNote: Each sample in this reference step uses 100 µ The working fluid of L can be adjusted by users according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer (component C): Dilute component C 10 times with ddH2O.(2) Configure 5 x Click iT EdU buffer additives (component E): add 300 µ Mix L of ddH2O into a 30 mg E component tube (final concentration of 100 mg/mL) until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio, and prepared into a 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 1.Table 1 Click it working fluidReaction componentsTaking the sample size of 10 holes as an example1×Click-iT EdU Reaction buffer855 µLCuSO4 (Component D)40 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Remove penetration enhancer, 100 per well µ Wash twice with 3% BSA washing solution of L.(6) Add 100 to each hole µ L Click iT working solution, evenly covering cells.(7) Incubate at room temperature in dark for 30 minutes.(8) Remove Click-iT working fluid and add 100 µ After washing cells twice with 3% BSA, remove the washing solution and add 100 µ L PBS keeps cells moist. If there are no other special requirements, photography analysis can be carried out.6. DNA re staining (optional)(1) Using 100 µ Wash the cells once with PBS and remove the washing solution.(2) Dilute Hoechst 33342 (component F) 2000 times with PBS.(3) Add 100 to each hole µ Incubate L 1 x Hoechst 33342 solution at room temperature in dark for 15-30 minutes.(4) Remove Hoechst 33342 solution and use 100 µ Wash cells twice with PBS.7. Imaging and analysisIt is recommended to take fluorescence microscopy photos immediately after staining is completed for observation; If conditions permit, please store in a dark and moist environment at 4 ° C for 3 days before taking photos. Flow cytometry detection method1. Cell cultureInoculate 1 × 105~3 × 106 cells per well into a 6-well plate.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU labeled cells(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours. The time of EdU incubation of cells can be directly used as an indicator for measuring cell DNA synthesis, and the choice of time point and incubation time depend on the cell growth rate. Pulse labeled cells incubated with brief EdU can be used to study cell cycle dynamics.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations such as 10-50 should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash cells twice with 1% BSA after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, collect cells, add 1 mL of PBS to each tube to clean the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to remove EdU residue that has not been added to DNA.(2) Add 1 mL of 4% paraformaldehyde fixative to each tube to resuspend cells.(3) Incubate at room temperature for 20 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(4) Add 1 mL of 2 mg/mL glycine to each tube and incubate for 5 minutes. Neutralize the remaining fixed solution, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of PBS to each tube for cleaning once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(5) Add 1mL of 0.5% Triton X-100 osmotic enhancer to each tube and resuspend cells. Incubate at room temperature for 10 minutes.5. EdU detectionNote: For 6-well plate samples, reference can be made to 1 mL of working solution per well. Users can adjust the dosage according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer: Dilute component C 10 times with ddH2O.(2) Prepare 5 x Click iT EdU buffer additives (component E): Add 300 µ L ddH2O to 30 mg of component E in a test tube (final concentration 100 mg/mL), mix well until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio to form 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive storage solution with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 2.Table 2 Click it working fluidReaction componentsVolume of liquid required for a single reaction1×Click-iT EdU Reaction buffer875 µLCuSO4 (Component D)20 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Soak at 1000 rpm for 5 minutes, discard the supernatant, remove the enhancer, add 1mL of 1% BSA washing solution to each tube and wash twice. Soak at 1000 rpm for 5 minutes, discard the supernatant.(6) Add 1 mL of Click iT working solution to each tube and mix well.(7) Incubate at room temperature in dark for 30 minutes.(8) Soak at 1000 rpm for 5 minutes, discard the staining reaction solution, add 1% BSA to each tube to wash the cells twice, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells again with 1 mL of 1% BSA (the volume of resuspend cells can be adjusted according to the number of cells), and detect with a flow cytometer.Note: If other biomarker tests are required, please refer to step 4.6. Intracellular antigen labeling (optional steps)(1) Add antibody working solution and mix well.(2) Under dark conditions, incubate antibodies at appropriate temperature and time.7. Flow detection and analysis:(1) It is recommended to conduct flow cytometry testing immediately after dyeing is completed; If conditions are limited, please store in a dark place at 4 ℃ for testing, but it should not exceed 3 days.(2) It is recommended to test the number of cells up to one million levels as much as possible. If the number of cells is small, the number of cells tested can be adjusted to 100000 levels starting from the experiment. For cases where the cell yield is too low (just to the level of ten thousand), it may not be conducive to making a flow chart. Therefore, the cleaning frequency in step 5 (8) can be appropriately reduced.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 3. click it edu buffer additive solution should be prepared and used immediately to ensure the best results. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Cell proliferation detection (cell imaging flow universal)... 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