| Description | Inquire | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | DescriptionCAR10 is a kit that contains a selection of 10 carbohydrates/sugars: Arabinose, Fructose, Galactose, Glucose, α-Lactose, Maltose, Mannose, Ribose, Sucrose and Xylose, which may be used for general research, as reagents or as reference compounds in analytical procedures | CFDASE cell proliferation and tracking detection kit is a kit for cell proliferation and tracking detection based on CFDA se. This kit is composed of CFDASE powder, solvent and staining buffer. CFDASE is a derivative of fluorescein diacetate (FDA), which has cell membrane permeability and CFDASE cell proliferation and tracking detection kit is a kit for cell proliferation and tracking detection based on CFDA se. This kit is composed of CFDASE powder, solvent and staining buffer. CFDASE is a derivative of fluorescein diacetate (FDA), which has cell membrane permeability and does not have fluorescence luminescence. When CFDASE penetrates the cell membrane into living cells, it can be catalysed by esterases in the cytosol to produce carboxyfluorescein succinimidyl ester (CFSE), which can emit strong green fluorescence, cannot penetrate the cell membrane, and can remain intact in the cell. CFSE can also spontaneously and irreversibly covalently bind to intracellular amino groups to couple to cellular proteins. Meanwhile, the excess and uncoupled CFDASE returned to the extracellular medium by passive diffusion and was cleared by subsequent washing steps. The fluorescence of non dividing cells labeled by CFDASE is very stable, and the stable labeling time can reach several months, so it is very suitable for cell community analysis. The fluorescence of CFDASE labeled cells is very homogeneous, which is superior to other cell tracking fluorescent probes used previously, such as PKH26, and the fluorescence distribution of the divided progeny cells is also very uniform. In the process of cell division and proliferation, CFSE labeled fluorescence can be evenly distributed to the two progeny cells, and the fluorescence intensity becomes half of the parental cells. According to the fluorescence intensity, flow cytometer (FL1 channel) can detect undivided cells, cells that divide once (1 / 2 of the fluorescence intensity), twice (1 / 4 of the fluorescence intensity), three times (1 / 8 of the fluorescence intensity), and cells that divide more times. CFDASE can detect up to eight or more cleavages. CFDASE labeled cells can be used for proliferation studies in vitro and in vivo, and have the function of not staining adjacent cells. CFDASE is most commonly used to detect the proliferation of lymphocytes, and can also be used to detect the proliferation of fibroblasts, NK cells and other cells. CFDASE labeled cells showed green fluorescence. In addition to flow cytometry to detect cell proliferation, fluorescence microscopy can also be used for homogeneous staining of cell tracking observation.Components:ComponentsC598182-20TC598182-500TA. CFDA SE1 tube1 tubex5B.CFDA SE solvent20 µL500 µLC.10x CFDA SE Buffer1 mL x250 mLMatters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. CFDA and Se are easily hydrolyzed and will deteriorate quickly in aqueous solution. Please avoid contact with water during use. Contact with water during the process of labeling cells is within the permitted range. 3. CFDA se solvent will solidify at lower temperatures such as 4 º C and ice bath and stick to the bottom, wall or cover of the centrifugal tube. It can be used after incubating in a 20-25 º C water bath for a while until it is completely dissolved. 4. this kit optimizes the CFDA se staining system, but users are advised to explore the optimal working concentration and staining time according to their own cell type, culture conditions and application direction. Different cells have different lactonase activities, so the staining effect is different. 5. fluorescent dyes have quenching problems. Please avoid light during operation to slow down fluorescence quenching. 6. for your safety and health, please wear experimental clothes and disposable gloves.Usage method:1. Preparation of reagents(1) Preparation of CFDA SE storage solution: Take one tube of CFDA SE provided in the reagent kit and restore it to room temperature. Instantly centrifuge to allow the powder to fully settle to the bottom of the tube. Add 100 µ L CFDA SE solvent (add 20 µ L CFDA SE solvent) to it and dissolve it thoroughly to prepare CFDA SE storage solution (1000 ×). Prepared CFDA SE storage solution, stored at -20 ℃ in the dark, with a shelf life of two months- Storing at 70 ℃ in the dark can extend the usage time appropriately.(2) Preparation of CFDA SE Buffer: Dilute 10 x CFDA SE Buffer to 1 x with sterile cell culture grade water as needed. The prepared 1 × CFDA SE Buffer can be stored at 4 ℃ and can be stored at -20 ℃ if not in use for a long time.2. Marking and detection(1) Centrifuge the collected cells, use 1 mL 1 × CFDA SE Buffer to re suspend the cells in a 15 mL centrifuge tube, and adjust the cell concentration to 1-5 × 106 cells/mL.(2) Preparation of CFDA SE working solution: Dilute the CFDA SE storage solution (1000 ×) with 1 × CFDA SE Buffer to 2 ×.(3) Staining: Add 1 mL of CFDA SE working solution (2 x) to 1 mL of cell suspension to be labeled, invert and mix well, and incubate at 37 ℃ for 10 minutes.(4) Immediately add 5 times the volume of preheated complete culture medium (including serum) to the centrifuge tube, invert and mix well to terminate the labeling reaction.(5) Centrifuge at 1000 rpm for 5 minutes at room temperature to remove the supernatant, then wash once with 5-10 mL of complete culture medium.(6) Add 5-10 mL of complete culture medium and incubate at 37 ℃ for 5 minutes to promote the residence of CFDA SE in the cells and the entry of unreacted CFDA SE into the complete cell culture medium. Centrifuge at 1000 rpm for 5 minutes at room temperature to remove the supernatant and complete the final wash.(7) Subsequently, the cells can be cultured using the normal cultivation method. The labeling effect can be directly observed under a fluorescence microscope, or cell proliferation can be detected by flow cytometry after appropriate cultivation time, showing green fluorescence. The labeled cells can also be used for transplantation in live animals and for fluorescence tracing.Note: a If cell fixation is required, use aldehyde fixatives such as 4% paraformaldehyde to fix at room temperature for 15 minutes; If additional labeling such as antibody labeling is required afterwards, please permeabilize the cells with ice acetone for 10 minutes. b. The optimal labeling concentration and incubation time for CFDA SE vary for different cells. The initial experiment can be conducted according to the experimental steps. If the effect is not satisfactory, it is recommended to adjust the staining concentration and incubation time to achieve the best labeling effect.Scope of application：Cell proliferation assay... Read More | Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw cycleM665754DATP, 10 mM15 µL-20℃. Avoid freeze/thaw cycleM665754ERT Primer, 25 µM90 µL-20℃. Avoid freeze/thaw cycleM665754F5×SuperRT Buffer120 µL-20℃. Avoid freeze/thaw cycleM665754GUltraPure dNTP Mix, 10 mM each30 µL-20℃. Avoid freeze/thaw cycleM665754HSuperRT, 200 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754IRNase-Free Water1 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to give miRNA a Poly (A) tail, followed by reverse transcription using Oligo (dT) - Universal tag universal reverse transcription primers to synthesize the first stranded cDNA corresponding to miRNA. The miRNA cDNA first strand synthesis kit contains all the reagents required for the miRNA 3 'end Poly (A) tail modification process and the reverse transcription process after modification. This kit has a very high Poly (A) modification and reverse transcription efficiency, which can range from 1 ng-2 µ The first strand of cDNA corresponding to miRNA was effectively obtained from the total RNA of g. And the operation is simple and fast, which can be used to simultaneously detect multiple miRNAs from a synthesized cDNA reaction. This not only reduces errors and saves samples, but also achieves high-throughput detection.Note: This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit.Self prepared experimental materials: 1 ng-2 µ Total RNA of g, or 0.1 ng-1 µ Small molecule RNA of g.Notes:To prevent RNase pollution, attention should be paid to the following aspects:1. Use plastic products and gun heads without RNase to avoid cross contamination.2. Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use. Plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3. The solution should be prepared using water without RNase.4. Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.Usage:A. The process of miRNA adding Poly (A) tail:1.based on the amount of RNA used, dilute the total RNA of 10 mM ATP with 1 mM Tris (pH 8.0) according to the following formula: ATP dilution coefficient=5000/__ ngExample: If the initial amount of total RNA is 100 ng, then the ATP dilution coefficient is 5000/100=50. About to dilute ATP 50 times (1 µ 10 mM ATP plus 49 for l µ 1 mM Tris at pH 8.0.2. Add the following reagents to the pre cooled RNase free reaction tube in the ice bath to a total volume of 25 µ L. reagent 25 µlReaction system final concentration total RNA* X µl Up to 2 µg 10×Poly(A) Polymerase Buffer 2.5 µl 1× Diluted ATP in step "1" 1 µl / E. coli Poly(A) Polymerase, 5U/µl 0.5 µl 2.5 U RNase-Free Water up to 25 µl /*The total RNA used in the reaction must contain small molecule RNA.This process can also directly use small molecule RNA (recommended dosage of 2-5) µ L. Please determine the amount added based on the abundance of the target miRNA.3. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 37 ℃ for 15 minutes. After this process is completed, immediately proceed with the synthesis of the first strand cDNA or temporarily store it at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃.B. The process of synthesizing the first strand of modified miRNA cDNA:1. Add the reagents in the table below to the pre cooled RNase free reaction tube in the ice bath until the final volume reaches 20µl: reagent 20 µlReaction system The above Poly (A) reaction solution 4 µl UltraPure dNTP Mix ，10 mM each 1 µl RT Primer ，25 µM 3 µl 5×SuperRT Buffer 4 µl SuperRT ，200 U/µl 0.5 µl RNase-Free Water 7.5 µl2. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 42 ℃ for 50 minutes.3.85 ℃ for 5 minutes and terminate the reaction. The synthesized cDNA reaction solution can be directly used for fluorescence quantitative detection experiments or stored at -20 ℃ for future use... Read More |