| Description | Cell proliferation assays are widely used in the evaluation of cell viability, genotoxicity, and the efficacy of antitumor drugs. Direct detection of DNA synthesis in cells is considered the most accurate method for assessing cell proliferation. EdU (5-ethynyl-2′-deoxyuridine) is a Cell proliferation assays are widely used in the evaluation of cell viability, genotoxicity, and the efficacy of antitumor drugs. Direct detection of DNA synthesis in cells is considered the most accurate method for assessing cell proliferation. EdU (5-ethynyl-2′-deoxyuridine) is a novel thymidine (thymine deoxyribonucleoside) analogue. During DNA synthesis, EdU can be incorporated into newly synthesized DNA in place of thymidine. The ethynyl group on EdU can undergo a covalent reaction with fluorescently labeled small-molecule azide probes (such as Azide Alexa Fluor 488, Azide Alexa Fluor 555, Azide Alexa Fluor 594, Azide Alexa Fluor 647, etc.) via Cu(I)-catalyzed click chemistry, forming a stable triazole ring. This reaction is highly efficient and is referred to as the Click reaction. Through this process, newly synthesized DNA is labeled with the corresponding fluorescent probes, enabling the detection of proliferating cells using appropriate fluorescence detection equipment.Product Component TableE1456506Component50 T100 T200 TStorage conditionsQuantity Per TestE1456506AEdU(10 mM)100µL200µL400µL-20℃.Store in the dark.2 µL per 1.0-2.0 × 10⁶ cellsE1456506B6-FAM125µL250µL500µL-20℃.Store in the dark.2.5 µL per 1.0-2.0 × 10⁶ cellsE1456506CClick Reaction Buffer13 mL26 mL52 mL-20℃.Store in the dark.237.5 µL per 1.0-2.0 × 10⁶ cellsE1456506DCuSO40.5 mL1 mL2 mL-20℃.10 µL per 1.0-2.0 × 10⁶ cellsE1456506EClick Additive248 mg496 mg992 mg-20℃.Store in the dark.250 µL per 1.0-2.0 × 10⁶ cellsE1456506FDAPI Staining Solution（1000×）25 µL50 µL100µL-20℃.Store in the dark.0.5 µL per 1.0-2.0 × 10⁶ cells Usage Protocol1. Preparation1) Preparation of Click Additive Solution: For a 50-test kit: Add 12.5 mL of pre-chilled deionized water to the tube. Mix thoroughly until completely dissolved to obtain the Click Additive Solution. For a 100-test kit: Add 25 mL of pre-chilled deionized water to the tube. Mix thoroughly until completely dissolved to obtain the Click Additive Solution. For a 200-test kit: Add 50 mL of pre-chilled deionized water to the tube. Mix thoroughly until completely dissolved to obtain the Click Additive Solution. After preparation, aliquot the solution as needed and store at -20°C. If a white precipitate forms after dissolution, invert the tube repeatedly until it is fully dissolved before use. If the solution turns brown, it indicates degradation of the active component; discard it.2) Upon initial dissolution of the Click Reaction Buffer, aliquot it according to the number of samples per experiment and store at -20°C.2. EdU Labeling of CellsIt is recommended to use a final EdU concentration of 10 µM (1×). A 1:500 dilution of EdU (10 mM) in cell culture medium yields a 2× EdU working solution (20 µM). Mix an equal volume of pre-warmed (37°C) 2× EdU working solution (20 µM) with the cell suspension to achieve a final 1× EdU concentration. Incubate in a 37°C, 5% CO₂ incubator. Factors such as cell culture medium, cell density, cell type, and other experimental conditions may affect labeling efficiency. Therefore, the optimal EdU concentration and labeling duration must be empirically determined based on the cell type under investigation.3. Fixation and Permeabilization1) Harvest cells and centrifuge at 300 ×g for 5 min. Wash cells twice with PBS containing 2% FBS.2) Fix cells with 4% paraformaldehyde solution. Mix thoroughly and incubate for 15 min at room temperature protected from light.3) Collect cells and centrifuge at 300 × g for 5 min. Wash cells twice.4) Resuspend cells in PBS containing 0.1% Triton X-100. Mix well and incubate for 15 min at room temperature.5) Centrifuge at 300 × g for 5 min and wash cells twice.4. Fluorescent Labeling1) This protocol is based on a 500 µL reaction system per 2 × 10⁶ cells. The volume of the Click reaction mixture can be adjusted according to the experimental sample size.2) Centrifuge the cells at 300 ×g for 5 minutes. Add 500 µL of Click reaction mixture per sample, mix gently, and incubate for 30 minutes at room temperature protected from light.3) After the reaction, wash the cells twice with PBS containing 2% FBS.4) Dilute the DAPI Staining Solution (1000×) to 1× using PBS containing 2% FBS. Add 250 µL of the diluted DAPI solution to each sample and incubate for 5 minutes at room temperature.5) Add an additional 250 µL of PBS containing 2% FBS, mix gently, and proceed to detection using an appropriate flow cytometry instrument.Precautions1. Strictly adhere to the component order and volumes specified in the table above when preparing the Click reaction mixture, as deviations may affect subsequent experimental results.2. The Click reaction mixture must be used within 15 minutes of preparation.3. To avoid fluorescence quenching, perform detection as soon as possible after sample preparation... Read More | The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry.The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry. Compared with other fluorescent substrates or fluorescent inhibitors of Caspase based on ( FLICA ) analysis, aladdin 488 Caspase-3 Substrate does not inhibit the apoptosis process of intact cells while detecting Caspase-3 activity. Substrate is composed of fluorescent DNA dyes coupled with Caspase-3 DEVD recognition sequence. Substrate initially had no fluorescence and entered the cytoplasm through the cell membrane. In apoptotic cells, Caspase-3 cleaves the Substrate and releases high-affinity DNA staining, which migrates to the nucleus to label DNA and emits bright green fluorescence.Therefore, aladdin 488 Caspase-3 Substrate is bifunctional, which can not only detect Caspase-3 activity, but also visualize the morphological changes of the nucleus during apoptosis. Aladdin 488 staining can be fixed in formaldehyde and compatible with subsequent immunostaining experiments.Parameters:aladdin 488：Ex/Em = 500/530 nm (with DNA)Component：Points for attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments. 2.Cells can be co-stained with a final concentration of 1µM Hoechst 33342 dye to produce blue fluorescence staining of the nucleus ( Ex / Em = 346 / 460 nm ). 3.Aladdin 488 staining can be fixed by formaldehyde, but it is not compatible with methanol fixation. 4.Formaldehyde-fixed aladdin 488-stained cells can be treated with 0.1 % TritonX-100 for subsequent staining, but the brightness of the treated staining may be weakened. 5.Fluorescent dyes all have quenching problems, please try to avoid light to slow down the fluorescence quenching. 6.For your safety and health, please wear experimental clothes and wear disposable gloves.Scope of application：Caspase 3 kit and apoptosis detectionUsage:1. Experimental optimization: The experimental steps provided below are based on the endpoint detection system. Aladdin 488 Substrate can also be used for long-term cell incubation course research. Cell density, substrate concentration, and inhibitor concentration may need to be optimized. The optimal substrate concentration may be between 1-10 µ Between M. Cells can be incubated with substrates in culture medium, PBS, or other buffer of your choice. For adherent cells, we recommend replacing them with fresh culture media containing substrates to prevent background heterogeneity. The operation of changing the medium or washing the cells after substrate incubation is freely selectable.2. We suggest that you set the following controls:A. Negative control: cells that do not induce apoptosis;B. Positive control: cells that induce apoptosis;C. Inhibitor control: Induce cell apoptosis while incubating Caspase-3/7 inhibitors (or 10-30 minutes in advance), and finally add Aladdin 488 Caspase-3 substrate.3. The Caspase-3/7 inhibitor Ac-DEVD-CHO in the Ac-DEVD-CHO Caspase-3 inhibitor control kit can be used to confirm that Caspase-3/7 depends on the fluorescence signal of aladdin 488. For inhibitor control, the final concentration of the inhibitor should be at least twice the substrate concentration (e.g. when using 5 µ At substrate M aladdin 488, the concentration of Ac-DEVD-CHO is 10 µ M). Before adding the substrate, incubate Ac-DEVD-CHO at room temperature for 15-30 minutes. After adding the substrate, continue to retain the inhibitor in the incubation solution. Ac-DEVD-CHO is a reversible competitive inhibitor. In certain cell types, effective Caspase-3/7 inhibitors require the use of irreversible inhibitors, such as Z-DEVD-FMK, or the addition of inhibitors before or during apoptosis induction.4. Flow cytometry(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.(2) Adhering cells should be digested with trypsin or other methods before performing the aladdin 488 Caspase-3 experiment.(3) Resuspend cells with culture medium or buffer to achieve a cell density of 106 cells/mL(4) Suck 0.2 mL of cell suspension into a flow cytometry test tube.(5) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(6) 200 µ Add 5 to L cell suspension µ Substrate of 0.2 mM and immediately mix to achieve a substrate concentration of 5 µ M. The optimal substrate concentration for different cells may vary and requires analysis and optimization.(7) Incubate cells at room temperature in dark for 15-30 minutes.(8) Join 300 µ L-medium or PBS, analyzed by flow cytometry. Detect the channel for green fluorescence (Ex/Em=485/515 nm).5. Fluorescence microscope(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.(2) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(3) Using a solution containing 5 µ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.(4) Incubate cells at room temperature for 30 minutes or longer.(5) Cells can be directly observed in culture media containing Substrate. For the endpoint analysis method, PBS was used to clean the cells, fluorescence microscopy was used to observe the cells, and a filter (Ex/Em=485/515 nm) was used to observe green fluorescence.6. Fluorescence enzyme-linked immunosorbent assay (ELISA) reader(1) Adherent cells grow in black 96 well plates; Suspend cells, adjust the density to 106 cells/mL, and divide 0.2 mL of cell suspension into one well.(2) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls. Note: Cells may be processed in tubes or bottles and then transferred to a 96 well detection plate.(3) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(4) For suspended cells, directly add Substrate and mix well. For adherent cells, use a solution containing 5 µ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.(5) Cells can be directly observed in culture media containing Substrate.(6) For suspended cells, gently shake to resuspend the cells. The fluorescence enzyme-linked immunosorbent assay instrument is set with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Suggest using bottom collection method for adherent cells. Changes in the density of adherent cells may lead to inaccurate readings... Read More | 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 | 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 | Inquire |