| Description | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | 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 | Product introduction：This kit uses an improved SDS alkaline lysis method combined with DNA preparation membrane to selectively adsorb DNA to achieve the purpose of rapid purification of plasmid DNA. It is suitable for extracting up to 100u of high-purity plasmid DNA from 30-100 ml of Product introduction：This kit uses an improved SDS alkaline lysis method combined with DNA preparation membrane to selectively adsorb DNA to achieve the purpose of rapid purification of plasmid DNA. It is suitable for extracting up to 100u of high-purity plasmid DNA from 30-100 ml of bacterial culture for sequencing, in vitro transcription and translation, restriction enzyme digestion, bacterial transformation and other molecular biology experiments.Scope of application：Nucleic acid extraction and purification... Read More | Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no cytotoxicity in cell culture medium, so multiple downstream experiments can be performed using the same detection plate. CCK-8 method is a convenient colorimetric method for the determination of cell viability. It does not need the solubilization process and only needs the least steps to provide the results. The CCK-8 method can be used for the determination of 96-well microplates and high-throughput screening of 384-well microplates. Advantage：At present, the commercially available liquid CCK-8 kits generally have defects such as harsh storage conditions ( -4C or -20 ), unstable use in different pH ranges, and easy deterioration ( discoloration or precipitation ). The solid instant CCK-8 kit adopts a new formula and Swiss process, which overcomes these shortcomings of the liquid CCK-8 kit. It can be stored at room temperature for a long time ( > 3 years ), ready to use, stable in a wide pH range, and the experimental results are more reliable. Compared with the liquid CCK-8 kit, the solid-soluble CCK-8 kit has higher sensitivity and the biological response time is shortened by half.Application scope:It can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity test and activity detection of biological factors. Operating instructions:This reagent kit can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity assay, and activity detection of biological factors.1. Carefully and slowly tear along the gap in the packaging bag;2. Pour all the powder in the bag into a clean container containing 10mL of ultrapure water, shake continuously for 1 minute, and use it when the solid is completely dissolved;3. Unused reagents must be stored at low temperatures below 4 ℃.Equipment required for testing:Enzyme reader 96 well plate with 450-490 nm filter;Carbon dioxide incubator;96 well plate, sterilized transparent plate for cell detection;Multi channel pipette (8 or 12 channels: 10-100 µ l);Blood cell counter or cell counter.Cell viability testing:1. Inoculate cell suspension (100 µ l/well) into a 96 well plate and pre culture the plate in a carbon dioxide incubator for 24 hours (37 ℃, 5% CO2);2. Add 10 µ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as it may affect the reading of OD value);3. Incubate the culture plate in the incubator for 1-4 hours;4. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader;5. If the OD value is not determined temporarily, 10 µ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.Cell proliferation toxicity testing:1. Inoculate cell suspension (100 µ l/well) into a 96 well plate and pre culture the plate in an incubator for 24 hours (37 ℃, 5% CO2);2. Add 10ul of different concentrations of the substance to be tested to the culture plate;3. Incubate the culture plate in the incubator for an appropriate period of time (e.g. 6, 12, 24, or 48 hours);4. Add 10 µ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as they may affect the reading of the OD value);5. Incubate the culture plate in the incubator for 1-4 hours;6. Measure the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader;7. If the OD value is not determined temporarily, 10 µ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.Calculation method for cell survival rate/inhibition rate:Cell survival rate=[As Ab)/(Ac Ab)] x 100%Inhibition rate=[(Ac As)/(Ac Ab)] x 100%As: absorbance of experimental wells (including cells, culture medium, CCK-8 solution, and drug solution);Ac: absorbance of control wells (including cells, culture medium, CCK-8 solution, without drugs);Ab: Blank well absorbance (including culture medium and CCK-8 solution, excluding cells and drugs).Points for attention： 1.Unused reagents must be stored at low temperature below 4 °C, and stored in the dark at-20 °C for two years after unpacking, so as to avoid repeated thawing ; 2.The culture time of CCK-8 is generally 1-4 hours, but the naked eye can be taken out to observe the color degree in about 30 minutes. According to the cell type, the conditions need to be explored. The best reaction time of CCK-8 is based on the best time of specific color development.3. It is recommended to do a few holes to explore the number of inoculated cells and the culture time after adding CCK-8 reagent ; 3.The WST-8 in this kit will react with reducing agents ( such as some antioxidants ) to interfere with the detection. Before the cell proliferation-toxicity test, the background OD can be checked to confirm whether there is a reducing agent in the substance to be tested. If the effect of reducing agent needs to be removed, the fresh medium can be replaced before adding CCK-8 ( remove the medium, wash the cells twice with the medium, and then add the new medium ) ; 4.Phenol red in the medium does not affect the experimental results, and the absorbance of phenol red can be eliminated by deducting the absorbance of the background in the blank hole during calculation, so it will not affect the detection. 5.It is recommended to use a multi-channel pipette to reduce the difference between parallel holes. When adding CCK-8 reagent, it is recommended to add it obliquely to the wall of the culture plate, not to insert it under the liquid surface of the medium, which is easy to produce bubbles and interfere with OD determination. 6.If the drug contains metal, it has an effect on the color of CCK-8. The final concentration of 1mM lead chloride, ferric chloride and copper sulfate will inhibit the color reaction of 5 %, 15 % and 90 %, and reduce the sensitivity. If the final concentration is 10mM, the color reaction will be 100 % inhibited ; 7.When using a 96-well plate for detection, if the cell culture time is long, attention should be paid to the evaporation problem. On the one hand, because a circle around the 96-well plate is the easiest to evaporate, the method of discarding the surrounding circle can be adopted, and the same amount of PBS, water or culture medium can be added. On the other hand, the 96-well plate can be placed near the water source in the incubator to alleviate evaporation ; 8.When using standard 96-well plates, the minimum inoculation amount of adherent cells is at least 1,000 cells / well ( 100µl medium ). The sensitivity of detecting white blood cells is relatively low, so it is recommended that the inoculation amount should not be less than 2,500 cells / well ( 100 µl medium ). If you want to use a 24-well plate or a 6-well plate experiment, first calculate the corresponding inoculation amount per well, and add the CCK-8 solution according to 10 % of the total volume of the medium per well ; 9.Cell culture time varies according to the type and number of cells ( per well ), usually the color of white blood cells is weak, requiring a longer culture time ( 4 hours ) and a large number of cells ( ~ 105 cells / well ) ; 10.CCK-8 reagent is very low toxic to cells. The continuous reaction between it and dehydrogenase in living cells makes the color of the solution deepen and the OD value increase. The following methods can terminate the CCK-8 reaction ( 96-well plate ) : a ) After the color reaction, the culture plate was placed in a refrigerator at 4 ° C ; b ) 10µL 0.1MHCL solution was added to each well ; c ) 10 µL 1 % ( w / v ) SDS ( sodium dodecyl sulfate ) solution was added to each well. After the reaction stopped, the OD value should be measured within 24 hours. 11.To determine the specific number of cells, it is recommended to do the standard curve at the same time... Read More |