| Description | The Hoechst33258/PI Apoptosis Assay Kit is a detection kit that uses a dual fluorescence staining method with Hoechst 33258 and Propidium Iodide (PI) to analyze cell cycle and cell necrosis. While PI staining alone can observe the sub-G1 peak of apoptotic cells on the DNA histogram, it can only The Hoechst33258/PI Apoptosis Assay Kit is a detection kit that uses a dual fluorescence staining method with Hoechst 33258 and Propidium Iodide (PI) to analyze cell cycle and cell necrosis. While PI staining alone can observe the sub-G1 peak of apoptotic cells on the DNA histogram, it can only represent apoptosis in the G0/G1 phase and cannot observe apoptosis in the S and G2 phases. Moreover, after fixation, it is impossible to distinguish between live and dead cells. Hoechst 33258 can penetrate the cell membrane and bind to DNA in both normal and apoptotic cells, showing blue fluorescence under ultraviolet light. After staining, the fluorescence of apoptotic cells is significantly enhanced compared to normal cells. PI cannot penetrate the cell membrane and cannot stain normal or apoptotic cells with intact cell membranes. However, for necrotic cells, where the integrity of the cell membrane is lost, PI can penetrate the cell membrane and color the necrotic cells to produce red fluorescence. When these two dyes are used for double staining, normal cells appear weak blue, apoptotic cells appear bright blue, and necrotic cells appear bright blue and red.ComponentH1492197Component100TStorageQuantity Per TestH1492197AHoechst 33258 Staining Solution1 mL-20℃, Store in the dark.10 µL per 0.5-1.0x10⁶ H1492197BPropidium iodide Staining Solution (PI)0.5 mL-20℃, Store in the dark.5 µL per 0.5-1.0x10⁶ Note: The recommended number of cells to stain per test 0.5–1.0×10⁶ cells.Usage method:1. Cell PreparationAdherent cells: Grow cells in 6-well plates to the logarithmic growth phase. Wash twice with PBS and add 1 mL of PBS.Suspension cells: Wash cells twice with pre-cooled PBS and resuspend in PBS at a density of 0.5–1.0×10⁶ cells/mL.2. StainingAdherent cells: Add 10 µL of Hoechst 33342 and 5 µL of PI directly to each well of the 6-well plate. Incubate at 4 °C in the dark for 20–30 minutes.Suspension cells: Take 1 mL of the cell suspension, add 10 µL of Hoechst 33342 and 5 µL of PI, and incubate at 4 °C in the dark for 20–30 minutes.Note: After staining, proceed with fluorescence detection as soon as possible. The number of cells for each detection should not exceed 1×10⁶.3. Fluorescence Microscopy Detection and AnalysisAdherent cells: Remove the staining solution, wash twice with PBS, add an appropriate amount of PBS, and observe under a fluorescence microscope.Suspension cells: Wash twice with PBS, resuspend the cells in PBS, transfer to a culture dish, and observe for red and blue fluorescence.Matters needing attention:1. After the staining process is completed, the detection should be carried out as soon as possible.Hoechst 33258 and PI are harmful to the human body. Please take protective measures when using them.2. For your safety and health, please wear a laboratory coat and put on disposable gloves when operating.3. This product is exclusively for scientific research purposes and must not be used for clinical diagnosis or treatment... 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 | D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H Buffer EB 4 mL RT D665729I Buffer PS 10 mL RT D665729J Spin Columns DF 50 Pcs 2-8 ℃ D665729K Collection Tubes 50 Pcs RTProduct Introduction:The basic principle of this reagent kit is that after DNA is treated with sodium bisulfite, unmethylated cytosine can be transformed into uracil, while methylated cytosine remains unchanged. And adopting an innovative high-temperature treatment method, the transformation time is greatly shortened, the transformation efficiency is improved, and the transformation efficiency can reach over 99%. At the same time, using a silicon-based membrane purification column, DNA can be recovered and purified from the methylated solution through a simple binding washing elution step. The recovered DNA has high purity and good integrity, and can be directly used for sequencing, methylated PCR detection, chip analysis, connection and transformation, enzyme digestion, labeling, microinjection, PCR and in vitro transcription and other molecular biology experiments.Self prepared reagents: anhydrous ethanol, 75% ethanol.Preparation and important precautions before the experiment1. Product usage method:(1) 10 times packaging preparation method: CT Conversion Agent is a solid mixture that must be prepared before first use. Add 2 ml sterile water and 100 µ M-Dissolving Buffer and 300 µ Add M-Diffusion Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 10 DNA treatments. In order to achieve better results, the prepared CT Conversion Agent should be used immediately. If not used immediately, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.(2) 50 times packaging preparation method: CT Conversion Agent and M-Dissolving Buffer are solid mixtures that must be prepared before first use. Add 5 ml of sterile water to the M-Dissolving Buffer and shake to dissolve. After all the solids have dissolved, transfer all the solution from the M-Dissolving Buffer tube to the CT Conversion Agent tube and add 5.5 ml of sterile water. Add 1.5 ml of M-Dilution Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 50 DNA treatments. In order to achieve better results, the CT Conversion Agent should be used immediately after preparation. If not immediately used, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.2. Before the first use, anhydrous ethanol should be added to the M-Wash Buffer according to the instructions on the reagent bottle label.Operation stepsThe range of DNA prepared each time is 1 ng-4 µ Between g, the optimal amount is 500 ng-2 µ G.1. Take 20 µ Add DNA sample into centrifuge tube (self provided), and if the sample amount is insufficient, replenish with water up to 20 µ L.2. Add 2.2 to the DNA sample µ Mix the sample well with the M-Dilution Buffer of l.3.42 ℃ water bath for 30 minutes.4. Add 220 to the sample obtained from the previous step µ Prepare the CT Conversion Agent solution, mix well, and incubate in an 80 ℃ constant temperature water bath in a dark place for 60 minutes.5. Add 480 to the solution in the previous step µ M - Buffer PA, gently mix upside down.6. Column balance: Add 200 to the spin columns DS that have been loaded into the collection tube µ Centrifuge at 12000 rpm (~13400 × g) for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7.Add all the solution obtained from step 5 to the adsorption column (already loaded into the collection tube), let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Attention: The maximum capacity of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.8. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute using M-Buffer PA, discard the waste liquid from the collection tube, and place the adsorption column in the recovery tube.9. Add 650 to the adsorption column µ M-Wash Buffer (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.10.12000 rpm for 2 minutes, discard the waste liquid, and place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).11. Place the adsorption column into a new centrifuge tube (provided by oneself), and add 20 drops to the middle position of the adsorption membrane in the air µ M-Elution Buffer (pH 8.5), leave at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 1 minute.12. Collect 20 µ Add 2.2 to DNA µ M-Diffusion Buffer, let it stand at room temperature for 30 minutes.13. Add 500 to the solution µ After pre cooling anhydrous ethanol, invert and mix well, and place the solution at -20 ℃ to precipitate for 30 minutes (overnight precipitation is more effective).14.12000 rpm for 15 minutes and gently discard the supernatant.15. Add 75% ethanol, centrifuge at 12000 rpm for 1 minute, pour out the supernatant, wait for ethanol to evaporate at room temperature, then add 20 µ Dissolve the M-Elution buffer and store the DNA at -20 ℃. The DNA collected in this step can be used for subsequent related experiments... Read More | Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly luciferase is widely used in gene regulation and drug screening. Firefly luciferase is a protein with a molecular weight of about 61 KD. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. The optical signal of this kit is a kind of instantaneous light, which needs to be detected immediately after adding the working solution. The half-life of optical signal is about 5 min.Instruction：1.Working fluid configuration ( 1 ) Restore all components to room temperature. ( 2 ) The component B ( stock solution ) was fully diluted with component A to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the dosage of a single experiment is small, it is recommended to subpackage according to a single dosage. At room temperature, the activity decreased by about 10 % after the working solution was configured for 3 h, and the activity decreased by about 25 % after 5 h. 2.chemiluminescence value detection ( 1 ) The cell culture plate was taken out from the incubator and incubated at room temperature for 20 min to restore it to room temperature ( 22-25 ° C ). ( 2 ) Add the same volume of firefly luciferase working solution with the medium to the culture plate and mix well. ( 3 ) Incubation at room temperature for 5 min. Note : The incubation time can be adjusted according to cell type and cell number. ( 4 ) The values were read by multifunctional microplate reader or chemiluminescence instrument ( instrument parameters : the determination time was 10 s, the determination interval was 2 s ).Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 3. to prevent interference between holes, it is recommended to use white opaque orifice plate.Recommendation：Component B is recommended to use sterile water in advance to configure 2 mg / mL storage solution, A component and B component configured as storage solution, and small batch packaging according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing. Component：One-Step Firefly Luciferase Assay Buffer；D-Luciferin Scope of application：Mainly used for ADCC detection... Read More | This reagent kit is specially developed for one-step RT-PCR experiments. Reverse transcription and PCR are carried out in the same reaction system, without the need to add reagents or open the tube cap during the reaction process, which improves detection sensitivity and experimental efficiency This reagent kit is specially developed for one-step RT-PCR experiments. Reverse transcription and PCR are carried out in the same reaction system, without the need to add reagents or open the tube cap during the reaction process, which improves detection sensitivity and experimental efficiency while avoiding contamination. This kit includes a brand new high-efficiency reverse transcriptase, a fast hot start DNA polymerase, as well as reaction buffer suitable for reverse transcription and PCR amplification, and other components necessary for the experiment. The loss of activity of SuperRT reverse transcriptase RNase H reduces RNA degradation in reverse transcription reactions. This reverse enzyme has high reverse transcription efficiency and can perform good reverse transcription reactions on a small amount of RNA templates. The rapid hot start DNA polymerase used in PCR reaction has excellent performance of high amplification efficiency, strong specificity, and fast extension speed. The unique buffering system maximizes the efficiency of both reverse transcriptase and polymerase. The target product amplified using this reagent kit has an A base attached to the 3 'end, which can be directly used for T/A cloning.S665660Component100 TStorageS665660ASuperRT OneStep EnzymeMix50 µL-20℃. Avoid freeze/thaw cycle.S665660B2×SuperRT OneStep Buffer1.4 mL-20℃. Avoid freeze/thaw cycle.S665660CRNase-Free Water1.5 mL-20℃. Avoid freeze/thaw cycle. Notes:1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.3. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.4. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT-PCR reactions. When designing primers, factors such as GC content, primer length, primer position, and the secondary structure of PCR products need to be considered. It is recommended to use professional primer design software.Usage:1. Dissolve the RNA template, primers, OneStep RT-PCR Buffer, SuperRT OneStep RT-PCR EnzymeMix, and RNase Free Water and place them on ice for later use.2. Prepare the reaction system according to the following table: Reagent 25 µlReaction system Final concentration 2×SuperRT OneStep Buffer 12.5 µl 1× Forward Primer，10 µM 1 µl 0.4 µM Reverse Primer，10 µM 1 µl 0.4 µM SuperRT OneStep EnzymeMix 0.5 µl / RNA Template X µl 1 pg – 1 µg RNase-Free Water up to 25 µl / Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.3. Vortex and shake well, centrifuge briefly, and collect the solution to the bottom of the tube.4. Preheat the thermal cycler to 45 ℃, place the PCR tube in the thermal cycler, and perform RT-PCR reaction.Reaction conditions: Step Temperature Time / Reverse transcription 45℃ 30 min / PCR pre denaturation 95℃ 2 min Denaturation 94℃ 30 s 30-40 cycles Anneal 55-65℃ 30 s 30-40 cycles Extend 72℃ 30 s 30-40 cycles Finally extended 72℃ 5 min /Attention:1) In general PCR experiments, the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the annealing time is generally 20-30 seconds. If the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time is set based on the size of the amplified fragments, and the DNA Polymerase amplification efficiency contained in this product is 1 kb/30s.3) The number of cycles can be set based on the downstream application of the amplification product. Too few cycles, insufficient amplification; Multiple cycles increase the probability of mismatches and result in severe non-specific backgrounds. Therefore, while ensuring product yield, the number of cycles should be minimized as much as possible.5. After the reaction is complete, take 5 µ l of the reaction product, add an appropriate amount of loading buffer, and perform electrophoresis detection results... Read More |