| Description | The AO/PI Double Staining Kit is a detection system based on fluorescent nucleic acid dyes, designed to quickly and reliably distinguish live, apoptotic, and necrotic cells in biological samples. Its core principle utilizes the differences in cell membrane permeability and the specific The AO/PI Double Staining Kit is a detection system based on fluorescent nucleic acid dyes, designed to quickly and reliably distinguish live, apoptotic, and necrotic cells in biological samples. Its core principle utilizes the differences in cell membrane permeability and the specific fluorescent reactions upon binding to nucleic acids of the two dyes, Acridine Orange (AO) and Propidium Iodide (PI). AO can penetrate the cell membranes of all cell types (live, apoptotic, and necrotic). After entering the cell, AO binds to nucleic acids, but its fluorescence color depends on the target: when it binds to double-stranded DNA (dsDNA) in the nucleus, it emits green fluorescence (Ex 488 nm, Em 530 nm); whereas when it binds to single-stranded DNA (ssDNA) or RNA, it emits red fluorescence (Em >640 nm). In contrast, PI cannot penetrate cells with intact cell membranes. It only enters late apoptotic and necrotic cells, whose cell membranes have been compromised, and binds to DNA to produce red fluorescence (Ex 535 nm, Em 617 nm). Therefore, after AO/PI double staining, observation under a fluorescence microscope allows clear differentiation of cells in different states: live cells display normal green fluorescence; early apoptotic cells show bright green speckled nuclear morphology (only AO enters); late apoptotic and necrotic cells appear orange-red or strong red fluorescence due to the combined effect of AO and PI.Product InformationA1492198Components100T500TStorageQuantity Per TestA1492198AAO Staining Solution100µL500µL2-8℃, Store in the dark.1µL per 0.5-1.0 x 10⁶ cellsA1492198BPI Staining Solution500µL2500µL-20℃. Store in the dark.5µL per 0.5-1.0 x 10⁶ cellsA1492198CAO/PI Dilution Buffer10mL50mL2-8℃0.1mL per 0.5-1.0 x 10⁶ cellsNote: The recommended number of cells to stain per test is 0.5-1.0 x 10⁶ cells.Procedure1. Preparation of Working Solution(1) Take out the reagents and allow them to reach room temperature. Dilute the AO/PI Dilution Buffer 10-fold with double-distilled water before use.(2) Prepare the AO/PI Staining Working Solution: The concentration of AO stock solution in this product is 10 mg/mL, and PI stock solution is 1 mg/mL. Add 50 µL of PI stock solution and 10 µL of AO stock solution to 10 mL of diluted AO/PI Dilution Buffer. Vortex to mix well, resulting in 10 mL of AO/PI Staining Working Solution.Note: Since optimal staining conditions may vary with cell type, it is recommended that users determine the appropriate dye concentration individually based on practical conditions.2. Staininga. For Adherent Cells:(a) Seed cells in culture dishes, multi-well plates, or on coverslips, and treat the cells as required by the experimental design. Gently aspirate the medium from the wells and wash with PBS for about 10 seconds, then remove the PBS.(b) Add an appropriate volume of AO/PI Staining Working Solution, and gently swirl to ensure the dye evenly covers all cells.Note: For adherent cells cultured in 6-well plates with a confluency over 80%, it is recommended to add 1 mL of staining working solution per well. This can be optimized according to the specific experimental setup.(c) Incubate the cells at room temperature, protected from light, for 1-10 minutes.(d) Aspirate the staining solution, wash 2-3 times with PBS, then add serum-free culture medium. Observe immediately under a fluorescence microscope: observe AO-DNA green fluorescence at Ex/Em = 488/530 nm, and PI-DNA red fluorescence at Ex/Em = 535/617 nm. Alternatively, analysis by flow cytometry or detection with a fluorescence microplate reader can be performed after staining.b. For Suspension Cells:(a) After treating the cells as required by the experimental design, count the cells. Take an appropriate number of cells, centrifuge at 500 × g for 5 minutes at room temperature, gently aspirate the medium, resuspend in an appropriate amount of PBS, and centrifuge again at 500 × g for 5 minutes at room temperature to remove the PBS.(b) Add an appropriate amount of AO/PI Staining Working Solution to achieve a cell density of approximately 10⁶ cells/mL.(c) Incubate the cells at room temperature, protected from light, for 1-10 minutes.(d) Place a drop directly on a glass slide, cover with a coverslip, and observe under a microscope: observe AO-DNA green fluorescence at Ex/Em = 488/530 nm, and PI-DNA red fluorescence at Ex/Em = 535/617 nm. Alternatively, analysis by flow cytometry or detection with a fluorescence microplate reader can be performed after staining.Note: If background interference is significant, centrifuge to remove the staining solution, wash 1-2 times with PBS, and then observe under the microscope.Precautions1. AO/PI staining solutions are somewhat toxic; handle with care. 2. For your safety and health, please wear a lab coat and disposable gloves.3. Fluorescent dyes are subject to quenching; it is recommended to complete detection on the same day after staining whenever possible... Read More | DescriptionRefer to the product′s Certificate of Analysis for more information on a suitable instrument technique. Contact Technical Service for further support | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | V669947 Component 50T Storage V669947A Buffer GL 15 mL RT V669947B Buffer GW1 (concentrate) 13 mL RT V669947C Buffer GW2 (concentrate) 15 mL RT V669947D Buffer RE 10 mL RT V669947E Proteinase K 12.5 mg RT V669947F Proteinase K Storage Buffer 1.25 mL RT V669947G Spin Columns RS with Collection Tubes V669947 Component 50T Storage V669947A Buffer GL 15 mL RT V669947B Buffer GW1 (concentrate) 13 mL RT V669947C Buffer GW2 (concentrate) 15 mL RT V669947D Buffer RE 10 mL RT V669947E Proteinase K 12.5 mg RT V669947F Proteinase K Storage Buffer 1.25 mL RT V669947G Spin Columns RS with Collection Tubes 50 RT V669947H RNase-Free Centrifuge Tubes (1.5 mL) 50 RTProductsThis kit is suitable for the extraction of viral RNA and DNA from fresh or frozen plasma, serum and cell-free body fluids. It is easy to operate as it does not require the use of organic solvents such as phenol and chloroform for extraction. The kit uses a unique buffer system to enable efficient and specific binding of viral nucleic acids in lysate to silica gel centrifugal adsorption columns. Inhibitors of PCR and enzyme reactions as well as residual impurities can be efficiently removed in a two-step effective rinsing step, and finally high purity viral nucleic acids can be obtained by using a low-salt buffer or water for elution. The purified viral nucleic acid is free of protein, nuclease and other impurities, and can be used directly in PCR, RT-PCR, Real-Time PCR, blotting experiments and so on.Self-contained reagent: anhydrous ethanol.Pre-experiment 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. Do not add Proteinase K directly into Buffer GL.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. Avoid repeated freezing and thawing of serum or plasma, which can lead to protein denaturation or precipitation, reducing the viral titer and thus affecting the yield of extracted viral nucleic acids.4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the label instructions of the reagent bottle before first use.5. Check Buffer GL for crystallization or precipitation before use. If crystallization or precipitation occurs, redissolve Buffer GL in a water bath at 56℃.Procedure1. Take a 1.5 ml centrifuge tube (self-provided) and add 20 µl Proteinase K.2. Add 200 µl serum or plasma to the centrifuge tube. Add 200µl Buffer GL and vortex and shake for 15 seconds.Note: 1) Sample volume less than 200 µl can be made up by adding 0.9% NaCl (self-provided). 2) In order to ensure effective lysis of the sample, the sample needs to be mixed well with Buffer GL after adding Buffer GL.3. Incubate at 56°C for 15 minutes, centrifuge briefly, and collect the solution from the wall of the tube to the bottom of the tube.4. 250 µl of anhydrous ethanol was added, vortexed and shaken for 15 seconds, left at room temperature for 5 minutes, centrifuged briefly, and the solution on the wall of the tube was collected at the bottom of the tube.Note: If the ambient temperature exceeds 25°C, anhydrous ethanol should be used after pre-cooling on ice.5. Add the solution obtained in step 4 to the adsorbent column (RNase-Free Columns RS) that has been loaded into the collection tube, and if the solution cannot be added at one time, it can be transferred in several times. centrifuge the column at 12,000 rpm (~13,400 × g) for 1 min, pour off the waste liquid in the collection tube, and put the column back into 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 put the adsorption column back into 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. Add 500 µl of anhydrous ethanol to the adsorbent column and centrifuge at 12,000 rpm for 1 min. Pour off the waste liquid in the collection tube and put the adsorbent column back into the collection tube.9. Centrifuge at 12,000 rpm for 3 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 the removal of residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).10. Place the adsorption column in a new collection tube (RNase-Free Centrifuge Tube), add 20-150 µl of Buffer RE or sterilized water overhanging the middle of the adsorption column membrane, leave it at room temperature for 2-5 minutes, and then centrifuge it at 12,000 rpm for 1 minute to collect the nucleic acid solution.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 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) For long-term storage, please store the DNA solution at -20℃ and the RNA solution at -70℃.3) If the final concentration of DNA/RNA is to be increased, the DNA/RNA eluate obtained in step 10 can be re-spiked onto the adsorbent membrane and step 10 repeated... Read More |