| Description | Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.Assay Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.Assay PrincipleACK catalyzes the synthesis of Acetyl Phosphate and ADP from Sodium Acetate and ATP. Pyruvate Kinase then catalyzes the conversion of ADP and Phosphoenolpyruvate (PEP) to ATP and Pyruvate. Subsequently, Lactate Dehydrogenase catalyzes the reduction of Pyruvate by NADH to produce Lactate and NAD⁺. The rate of oxidation of NADH to NAD⁺, measured by the decrease in absorbance at 340 nm, reflects ACK activity.Component48T96TStorageExtraction Buffer60 mL60 mL×22-8℃ReagentⅠ15 mL30 mL2-8℃ReagentⅡ1EA2EA-20℃. Store in the dark.Reagent III25 µL50 µLNote: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.Required Materials and Equipment (Not Provided)Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)96-well UV plate or micro quartz cuvetteAdjustable pipettes and tipsConstant temperature water bathIce makerCentrifugeDeionized waterHomogenizer (for tissue samples)Reagent PreparationExtraction Buffer: Ready-to-use. Equilibrate to room temperature (RT) before use. Store at 4°C.Caution: Extraction Buffer is toxic and has a pungent odor. It is recommended to handle it within a fume hood.Reagent Ⅰ: Ready-to-use. Equilibrate to RT before use. Store at 4°C.Working Reagent Ⅱ: Prepare immediately before use. For one vial of Reagent Ⅱ, add 11 mL of Reagent Ⅰ and 19.8 µL of Reagent III. Mix thoroughly to dissolve. Prepare fresh for each use. Can be stored protected from light at -20°C for one month.Reagent Ⅲ: Ready-to-use. Equilibrate to RT before use. Store at 4°C protected from light.Sample Preparation*Note: The use of fresh samples is recommended. If not used immediately, samples can be stored at -80°C for up to one month. Control the temperature and time during thawing. If thawed at room temperature, complete the process within 4 hours.*1.Tissues: Weigh approximately 0.1 g of sample. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 15,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.Cells or Bacteria: Collect 5 million cells or bacteria by centrifugation. Wash the pellet with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt the cells/bacteria by sonication on ice (200W power, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 15,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.3.Serum (Plasma) or other liquid samples: Assay directly. If the solution is turbid, centrifuge first and use the supernatant for assay.Note: To determine protein concentration, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.Assay Procedure1.Preheat the microplate reader or spectrophotometer for 30 min. Set the wavelength to 340 nm. Zero the spectrophotometer with deionized water.2.Pre-warm a sufficient volume of the prepared Working Reagent Ⅱ at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes. Use immediately.3.Assay Setup (perform in a 96-well UV plate or micro quartz cuvette):ReagentTest Well (µL)Sample20Working Reagent Ⅱ180Mix thoroughly immediately after addition. Measure the absorbance at 340 nm at 10 seconds (A₁) and again at 190 seconds (A₂). Calculate ΔA = A₁ - A₂.Note: It is advised to run a pilot test with 2-3 samples showing expected significant variation beforehand. If ΔA is less than 0.05, consider increasing the sample volume or extending the reaction time to 10 or 20 minutes before measurement. If ΔA is greater than 1.0, dilute the sample further with Extraction Buffer (multiply the result by the dilution factor) or reduce the amount of sample used for extraction.Result CalculationNote: Both the derived and simplified calculation formulas are provided and are equivalent. The simplified formulas (in bold) are recommended for final calculation.1. Calculation for 96-Well UV PlateGeneral Parameters for 96-Well Plate:ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cmd (Light path of 96-well plate) = 0.5 cmVₜₒₜₐₗ (Total reaction volume) = 0.0002 L (200 µL)Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.02 mL (20 µL)T (Reaction time) = 3 minVₛₐₘₚₗₑₜₒₜₐₗ (Total extraction volume) = 1 mLCpr (Sample protein concentration, mg/mL)W (Sample mass, g)500 (Cell/Bacteria count in millions: 5 × 10⁶)1.1 Based on Sample Protein Concentration:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.Calculation:ACK Activity (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ TSimplified Formula: ACK (U/mg prot) = 1072 × ΔA ÷ Cpr1.2 Based on Sample Mass:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh sample.Calculation:ACK Activity (U/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: ACK (U/g fresh weight) = 1072 × ΔA ÷ W1.3 Based on Bacterial or Cell Density:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells/bacteria in the reaction system.Calculation (for 5 million cells in 1 ml extract):ACK Activity (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: ACK (U/10⁴) = 2.144 × ΔA1.4 Based on Liquid Volume:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per milliliter of sample.Calculation:ACK Activity (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: ACK (U/mL) = 1072 × ΔA2. Calculation for Micro Quartz CuvetteUse the formulas above but adjust the light path *d* from 0.5 cm to 1.0 cm.Precautions1. Keep samples and all reagents on ice during the assay procedure to prevent denaturation and loss of activity.2. The temperature of the reaction mixture must be maintained at 37°C or 25°C. When using a cuvette, a small beaker filled with deionized water pre-warmed to 37°C or 25°C (placed in a water bath) can be used to hold the cuvette and maintain temperature during the reaction.3. This product is for scientific research use only. It is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets RTProductsThis kit is suitable for extracting high purity total DNA from Gram-negative and Gram-positive bacteria. 106-108 cells can be processed at a time, and up to 20 µg of total DNA can be obtained within one hour without the need for toxic solvents such as phenol or chloroform, and without the need for ethanol precipitation. The optimized buffer system enables the DNA in the lysate to be efficiently and specifically bound to the silica matrix centrifugal adsorption column, while other contaminants can flow through the membrane, and the inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step, and finally washed off with low-salt buffer or water, so that high-purity DNA can be obtained.The purified DNA can be used for downstream experiments such as digestion, PCR, Real-Time PCR, library construction, Southern Blot and molecular labeling, molecular labeling and other downstream experiments. Self-contained reagents: anhydrous ethanol; Enzymatic Lysis Buffer is required for extraction of Gram-positive bacteria.Enzymatic Lysis Buffer was prepared by 20 mM Tris, pH 8.0; 2 mM Na2-EDTA, pH 8.0; and 1.2% Triton X-100. 121°C sterilization for 20 minutes, and the appropriate amount of Lysozyme was added at a final concentration of 20 mg/ml. Pre-experiment Preparation and Important Notes1. Add 1.25ml Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. If extracting genomes from bacterial cultures with high accumulation of secondary metabolites or thick cell walls, it is recommended that samples be collected early in the logarithmic phase.4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.5. Before use, please check Buffer GTL and Buffer GL for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer GL and Buffer GTL in a 56℃ water bath.6. If the downstream experiments are sensitive to RNA contamination, 4µl of DNase-Free RNase A (100mg/ml) can be added before adding Buffer GL. RNase A is not provided in this kit.If the extracted samples are Gram-positive bacteria, customers need to prepare their own Enzymatic Lysis Buffer to treat the bacteria, which requires the use of Lysozyme (lysozyme) at a concentration of 20 mg/ml, which is not provided in this kit.Procedurei Extraction of genomic DNA from Gram-negative bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180 µl Buffer GTL to the precipitate and shake to resuspend the bacteria.3. Add 20 µl of Proteinase K, vortex and mix well, incubate at 56°C until the solution becomes clear, and invert or shake the centrifuge tube at intervals during the incubation to disperse the sample.Note: If RNA removal is required, add 4 µl of RNase A solution at a concentration of 100 mg/ml after the above steps are completed, shake to mix, and leave for 5-10 minutes at room temperature.4. Add 200µl Buffer GL and mix well with vortexing and shaking. Add 200µl of anhydrous ethanol and mix well with vortexing and shaking.Centrifuge briefly so that the solution on the walls of the tube collects at the bottom.Note: 1) If multiple samples are manipulated together, Buffer GL and anhydrous ethanol can be mixed in equal proportions and then added together, shaking to mix.2) The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in step 4 (including the precipitate formed) to the Spin Columns DM in the collection tube, or if the solution cannot be added all at once, transfer it several times. centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.6. Add 500 µl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and return the adsorption column to the collection tube.7. Add 500 µl of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorbent column at room temperature for several minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube, add 50-200 µl Buffer GE to the middle part of the adsorption column overhanging the center of the adsorption column, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 ℃. note: 1) If the downstream experiments are sensitive to the pH or EDTA, the elution can be done with sterilized water. The pH of the elution solution has a great influence on the elution efficiency. If water is used as the elution solution it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 9 can be re-spiked onto the adsorbent membrane and step 9 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃.i. Extraction of genomic DNA from Gram-positive bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180µl Enzymatic Lysis Buffer (self-provided) to resuspend the bacteria.Enzymatic Lysis Buffer is prepared as described in the Self-Prepared Reagents section in the front of the manual.3. Incubate at 37°C for 30 minutes.4. Add 20µl Proteinase K and mix well. Add 200µl of Buffer GL and mix well with vortexing and shaking.Note: Do not add Proteinase K directly to Buffer GL.Incubate at 5.56°C for 30 minutes.Note: 1) If desired, incubation at 95°C for 15 minutes will inactivate the pathogen, but 95°C incubation will cause some DNA degradation.(2) If RNA removal is required, add 4µl of RNase A solution at a concentration of 100mg/ml after the above steps are completed, shake and mix well, and leave for 5-10 minutes at room temperature.6. Add 200µl of anhydrous ethanol and mix well with vortex shaking.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.7. Add all of the solution obtained in step 6 (including the precipitate formed) to the Spin Columns DM that have been loaded into the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and put the column back into the collection tube.8. Add 500 µl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.9. Add 500 µl Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 9 can be repeated if further DNA purity is required.10. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).11. Place the adsorption column in a new centrifuge tube (self-provided), add 50-200 µl of Buffer GE to the center of the adsorption column overhanging the center of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 11 can be re-spiked onto the adsorbent membrane and step 11 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃... Read More | 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 | Inquire | Apoptosis refers to the cell autonomous and orderly death controlled by genes to maintain the stability of the internal environment. Apoptosis is different from cell necrosis. Apoptosis generally refers to a programmed cell death process that occurs during the development of body cells or under the Apoptosis refers to the cell autonomous and orderly death controlled by genes to maintain the stability of the internal environment. Apoptosis is different from cell necrosis. Apoptosis generally refers to a programmed cell death process that occurs during the development of body cells or under the action of some factors through the regulation of intracellular genes and their products. Cell necrosis is a cell death process that is caused by strong physical and chemical or biological factors to cause disordered changes in cells. The difference between apoptosis and necrosis lies in the characteristic morphological and biochemical changes, including the changes of cell membrane permeability and nuclear chromatin, the contraction of cytoplasm and the loss of membrane asymmetry. The oxazole yellow/pi membrane permeability apoptosis detection kit produced by our company is a dual fluorescence detection kit based on oxazole yellow and PI dyes. This kit is suitable for fluorescence microscopy, flow cytometry, fluorescence microplate reader and other fluorescence detection systems. Oxazole yellow is a non cell membrane penetrating cyanine monomer green fluorescent dye with high affinity for DNA. It basically has no fluorescence when it is not bound to DNA, but can emit bright green fluorescence after binding to DNA. When apoptosis occurs, the permeability of cell membrane changes. At this time, oxazole yellow can enter the cell and bind to DNA, emitting bright green fluorescence. Therefore, it is often used for the detection of apoptosis. It should be noted that oxazole yellow can also stain dead cells, so it needs to be double stained with PI that specifically fluorescently stains dead cells to effectively determine apoptosis. PI (propidium iodide) is a red fluorescent dye that can stain DNA. It is an analog of pyridine bromide that releases red fluorescence after embedding double stranded DNA. Although PI cannot pass through the membrane of living cells, it can cross the damaged cell membrane of dead cells to stain nuclei. Therefore, oxazole yellow combined with PI can be directly used for the detection of apoptosis. Apoptotic cells show green fluorescence, dead cells show both red and green fluorescence positive, and living cells have little or no fluorescence.Components: Components O598364-50T A. Oxazole yellow dye 50 µL B. Propidium Iodide (PI) 50 µLUsage (using flow cytometry as an example):1. Cell preparation(1) For adherent cells, after trypsin digestion, resuspend in culture medium and wash once with pre cooled PBS; The digestion time of trypsin should not be too long to prevent false positives. Note: Digest with trypsin and allow the cells to recover in the optimal cell culture conditions and medium for about 30 minutes, then stain.(2) For suspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash once with pre cooled PBS.2. Cell stainingSuspend cells in pre cooled PBS, with a recommended cell count of 106 cells/mL per sample. Add 1 µ L Oxazole Yellow and 1 µ L to 1 mL of the samplePI， Gently blow and mix well. Incubate on ice in the dark for 30 minutes. Note: We suggest adding the following two experimental controls:Blank tube: negative control group cells, without dye, used to regulate voltage.Single staining tube: Positive control group cells were treated with only two tubes, Oxazole yellow and PI, for regulating compensation.3. Flow detectionAfter incubation, the sample can be directly detected by flow cytometry, or centrifuged at 1000 rpm for 5 minutes, the supernatant can be aspirated, and the sample can be resuspended in 1 mL of pre cooled PBS for flow cytometry detection. Oxazole yellow can be excited by a 488 nm laser, and the detected fluorescence emission spectrum is around 530 ± 30 nm (FITC channel), while the PI channel emission spectrum is around 617 nm (PI or PE channel).Product parameters：Oxazole yellow dye:ex/em = 491 / 509 nm (bound DNA); Propidium iodine:ex/em = 535 / 617 nm (combined with DMatters 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 to slow down fluorescence quenching. 3. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Membrane permeability apoptosis assay... 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