| Description | Inquire | 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)... 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 | H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT Buffer 500 µL -20℃. Avoid freeze/thaw cycle. H665581E Primer Mix 120 µL -20℃. Avoid freeze/thaw cycle. H665581F RNase-Free Water 2×1 mL -20℃. Avoid freeze/thaw cycle.Product IntroductionThis product is a kit for removing genomic DNA for reverse transcription. The kit removes genomic DNA in 2 minutes at 42°C. Since the reverse transcription reagent contains a component that inhibits gDNA Eraser, cDNA can be synthesized directly by reverse transcription of gDNA Eraser-treated samples.The kit is equipped with a new high-efficiency reverse transcription enzyme, HiFiScript, with novel mutation sites that dramatically increase the transcriptional activity of the enzyme, resulting in higher efficiency and yield of cDNA first-strand synthesis. The first strand of cDNA can be synthesized with higher efficiency and yield, and the first strand of cDNA can be synthesized from pg total RNA or mRNA. If the reverse transcription product cDNA is used for downstream fluorescence quantitative detection, the reverse transcription reaction can be completed at 42℃ in 15 minutes. This kit is suitable for the synthesis of first-strand cDNA and subsequent RT-PCR, RT-qPCR, and the construction of full-length cDNA libraries.Product Features1. Rapid genome removal: contains gDNA Eraser for genomic DNA removal, which removes genomic DNA in just 2 minutes.2. Rapid reverse transcription: 15 minutes to obtain fluorescent quantitative PCR template cDNA first strand synthesis.3. High sensitivity: cDNA first strand can be synthesized using pg-level total RNA or mRNA templates.4. Highly efficient reverse transcription: Novel mutation sites dramatically increase enzyme activity, resulting in higher yields of cDNA.matters needing attention1. During operation, RNase contamination should be avoided to prevent RNA degradation or cross-contamination in the experiment. It is recommended that operators wear masks and disposable gloves and change the gloves frequently, and use specialized instruments and consumables.2. The reverse transcription system is prepared and operated on ice to prevent degradation of RNA. Store the kit enzymes at -20ºC as soon as possible after use and try to avoid repeated freezing and thawing.3. The reaction system can be scaled up to a maximum of 1 µg of total RNA in 10 µl of reaction system.4. Primer Mix is prepared by Oligo(dT) and Random primer, and Oligo-dT Primer or Gene Specific Primer can also be used according to the experimental needs.5. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).6. For RNA templates with complex secondary structures, it is recommended to incubate the template RNA at 65°C for 5 minutes immediately on ice prior to the manipulation step and centrifuge briefly before proceeding to the next step.UsageThaw template RNA on ice; place kit components on ice immediately after thawing at room temperature. Each solution was mixed by vortexing and shaking before use and briefly centrifuged.I. Genomic DNA removal reactions1. Prepare the reaction system according to the following table on ice in a total volume of 10 µl. To ensure the accuracy of the reaction solution preparation, prepare the premixed system in the amount of reaction number + 2 before dispensing it into each reaction tube and finally adding the RNA sample.Note: 1) If the amount of total RNA is greater than 1µg, scale up the reaction system proportionally. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).2. Mix by vortex shaking and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. Incubate at 42°C for 2 minutes (this can be extended to 30 minutes for room temperature reactions).4．At the end of the reaction, centrifuge briefly and place on ice to cool.II. Reverse transcription reaction1. Prepare the reaction system on ice according to the following table. In order to ensure the accuracy of the reaction solution configuration, first prepare a premixed solution in the amount of number + 2, and then dispense 10 µl into each reaction tube, take 10 µl of the prepared premixed solution and add it to the reaction tube of step 1 where the de-etching of the genome has been completed.Note: 1) Oligo-dT Primer or Gene Specific Primer can be used according to the needs of the experiment, it is recommended to use 50 pmol of Oligo-dT Primer or 2 pmol of Gene Specific Primer for 20 µl reaction system.2. Mix well and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. cDNA synthesis reaction conditions:1) If fluorescent quantitative PCR assay is performed downstream, incubate at 42°C for 15 minutes and 85°C for 5 minutes.2) If downstream for normal PCR assay, incubate at 42°C for 30-50 minutes and 85°C for 5 minutes. Note: For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance reverse transcription efficiency.4. At the end of the reaction, centrifuge briefly and place on ice before proceeding with subsequent PCR or fluorescence quantitative PCR, or place at -20°C if prolonged storage is required.Note: When performing Real-time PCR reactions, the amount of reverse transcription product added should not exceed 1/10 of the total volume of the PCR reaction... Read More | Product Content R669990Component50 TStorageR669990ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669990B10×Reaction Buffer1 mL-20℃. Avoid freeze/thaw cycle.R669990CBuffer RL35 mLRTR669990DBuffer RW135 mLRTR669990EBuffer RW2 (concentrate)11 mLRTR669990FRNase-Free Water10 Product Content R669990Component50 TStorageR669990ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669990B10×Reaction Buffer1 mL-20℃. Avoid freeze/thaw cycle.R669990CBuffer RL35 mLRTR669990DBuffer RW135 mLRTR669990EBuffer RW2 (concentrate)11 mLRTR669990FRNase-Free Water10 mLRTR669990GSpin Columns RM with Collection Tubes50 setsRTR669990HRNase-Free Centrifuge Tubes (1.5 mL)50 EART ProductsThis kit combines highly efficient guanidine isothiocyanate cleavage technology with silica matrix membrane purification for the efficient extraction of total RNA from animal cells and tissues, typically up to 30 mg of tissue or 1x107 cells as a starting sample. The kit also allows recovery of incompletely purified RNA, in vitro transcription and RNA from enzymatic reactions. high quality RNA with molecular weights greater than 200 bases can be extracted and purified using the kit with virtually no DNA residue. If RNA experiments that are very sensitive to trace DNA are to be performed, residual DNA can be removed by on-column digestion using RNase-free DNase. The extracted RNA can be used in downstream experiments such as RT-PCR, Nothern Blot and Dot Blot. Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction).Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.2) RNase-free water should be used to prepare the solution.(3) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the amount and quality of RNA extraction.3. Please add β-mercaptoethanol to Buffer RL before use, add 10µl of β-mercaptoethanol to 1ml of Buffer RL. Buffer RL with β-mercaptoethanol can be stored for 1 month at room temperature.4. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label.5. Buffer RL may be heated at 56°C to dissolve if precipitation occurs and then left at room temperature.All centrifugation steps are performed at room temperature and all maneuvers are performed quickly.Procedure1. Sample handling1a Tissue: Grind tissue in liquid nitrogen. Add 600 µl Buffer RL for every 20-30 mg of tissue (check for addition of β-mercaptoethanol before use), and 350 µl Buffer RL for tissue samples of less than 20 mg. Sample volume is not to exceed one-tenth of the Buffer RL volume.1b Cells in monolayer culture: Lysed or processed into cell suspension directly in culture flask, centrifuged to obtain cell precipitate, discarded the supernatant, added 600µl Buffer RL for every 6-10 cm2 of culture area, 350µl Buffer RL for less than 6cm2, and blown several times repeatedly to make the cells lysed sufficiently.1c Cell suspension: centrifuge at 12,000 rpm (~13,400 × g) for 1 min and discard the supernatant to obtain the cell precipitate. Add 600 µl Buffer RL for every 5×106-1×107 cells, and 350 µl Buffer RL for less than 5×106 cells, and blow several times repeatedly to fully lysate.Note: 1) Try to get rid of the cell culture medium, which may inhibit cell lysis affecting RNA yield.2) Try to keep the cells well suspended and well lysed, otherwise RNA yield is affected.2. After the sample is fully lysed, leave it at room temperature for 5 minutes to allow complete separation of the protein-nucleic acid complex.3. Centrifuge at 12,000 rpm for 2-5 min and remove the supernatant for the following operations.4. Add 1x volume (600µl or 350µl) of 70% ethanol (prepared without RNase water) to the solution obtained in step 3 and mix well.Note: The addition of ethanol may produce a precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in the previous step to the Spin Columns RM in the collection tube. If you cannot add all of the solution to the column at once, transfer it in two passes, centrifuge at 12,000 rpm for 1 minute, and discard the waste solution. Place the column back into the collection tube.Note: The maximum loading capacity of the adsorption column is 100µg, do not overload as this will affect the yield and purity of the RNA.6. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.7. Preparation of DNase I mixture: Take 52 µl of RNase-Free Water, add 8 µl of 10×Reaction Buffer and 20 µl of DNase I (1 U/µl) to it, mix well, and prepare a final volume of 80 µl of reaction solution.8. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.9. Add 200 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.10. Add 500µl Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge 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.11. Repeat step 10.12. 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 a few minutes to thoroughly dry the anhydrous ethanol in the adsorption column.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).13. Transfer the adsorbent column into a new centrifuge tube, add 30-50 µl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Wate should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 13 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 13 repeated... Read More |