| Description | Inquire | 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 | Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes;Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. ProcedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol.Index N501 Primer for IlluminaIndex N901-N996 Primer for Illumina... Read More | This kit combines efficient guanidine isothiocyanate lysis technology with silicon matrix membrane purification technology to efficiently extract total RNA from animal cells and tissues. The starting sample usually has a maximum of 30 mg of tissue or 1 x 107 cells. This reagent kit can also recover This kit combines efficient guanidine isothiocyanate lysis technology with silicon matrix membrane purification technology to efficiently extract total RNA from animal cells and tissues. The starting sample usually has a maximum of 30 mg of tissue or 1 x 107 cells. This reagent kit can also recover partially purified RNA, RNA obtained from in vitro transcription and enzymatic reactions. This reagent kit can extract and purify high-quality RNA with a molecular weight greater than 200 bases, with almost no DNA residue. If RNA experiments are to be conducted that are highly sensitive to trace amounts of DNA, residual DNA can be digested and removed on a column using DNase I without RNase. The extracted RNA can be used for downstream experiments such as RT-PCR, Northern Blot, Dot Blot, etc. R666020Component50 TStorageR666020ABuffer RL35 mLRTR666020BBuffer RW140 mLRTR666020CBuffer RW2 (concentrate)11 mLRTR666020DRNase-Free Water10 mLRTR666020ESpin Columns RM with Collection Tubes50 setsRTR666020FRNase-Free Centrifuge Tubes (1.5 mL)50 EART Self prepared reagents: β- Mercaptoethanol, anhydrous ethanol (newly opened or dedicated for RNA extraction).Preparation and important precautions before the experimentTo prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of RNA extraction.3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be heated at 56 ℃ and re solved. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1ml Buffer RL µ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.6. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase.Operation steps1. Sample processing1a organization: Grind the organization in liquid nitrogen. Add 600 to every 20-30 mg of tissue µ L Buffer RL (check if it is added before use) β- Mercaptoethanol), tissue sample less than 20 mg plus 350 µ Buffer RL. The sample volume shall not exceed one tenth of the buffer RL volume.1b Single layer culture of cells: The cells are directly lysed or processed into cell suspensions in a culture bottle, centrifuged to obtain cell precipitates, and the supernatant is discarded. 600 is added every 6-10 cm2 of culture area µ Buffer RL, less than 6 cm2, add 350 µ Blow buffer RL several times to fully crack it.1c cell suspension: Centrifuge at 12000 rpm (~13400 × g) for 1 minute to discard the supernatant and obtain cell precipitate. Add 600 cells every 5 × 106-1 × 107 cells µ Buffer RL, less than 5 × 106 cells added to 350 µ Blow buffer RL several times to fully crack it.Attention:1) Try to eliminate the cell culture medium as much as possible, as it may inhibit cell lysis and affect RNA production.2) Try to fully suspend and lyse the cells, otherwise it will affect RNA production.2. After the sample is fully lysed, it should be left at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Centrifuge at 2000rpm for 2-5 minutes, take the supernatant and proceed to the next step.4. Add 1 volume (600) µ L or 350 µ l) Mix 70% ethanol (prepared without RNase water) well.Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.5. Add all the solution obtained in step 4 to the Spin Columns RM that has been loaded into the collection tube. If it is not possible to add all the solution to the adsorption column at once, please transfer it in two batches, centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube. Attention: The maximum loading capacity of the adsorption column is 100 µ g, do not overload, otherwise it will affect the yield and purity of RNA.6. Add 700 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column in the collection tube.Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 6 with the following steps.1) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.2) Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1 U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.Attention: The above system is configured according to our company's DNase I reaction system. Please refer to the corresponding manual for other company products.3) Add 80 µ l of the prepared DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.4) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.7. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol is 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.8. Repeat step 7. 9. Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. 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 can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).10. Place the adsorption column in a new RNase free centrifuge tube, and add 30-50 to the middle of the adsorption column in the air µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 10 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 10... Read More | This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked andHomogenized samples, in their unique high salt state, RNA specifically binds to silicon matrix membranes, greatly reducingEffectively removing organic solvent contamination while removing protein contamination, resulting in higher purity and quality of RNA. bookThe product can quickly extract total RNA from various cells or tissues, and can process 30-50 mg of tissue or 5 × 10 ⁶ cells each time,Can handle multiple different samples simultaneously. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the residual DNA can be utilizedUsing DNase without RNase for digestion and removal on the column, the extracted RNA can be directly applied to RT-PCR Experiments such as Northern Blot, Dot Blot, and in vitro translation. U665516 Component 50 T Storage U665516A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. U665516B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. U665516C TRIzon Reagent 60 mL 2-8℃. Protect from light. U665516D TRIzon PaI™ 10 mL 2-8℃. Protect from light. U665516E Buffer RW1 40 mL RT U665516F Buffer RW2 (concentrate) 11 mL RT U665516G RNase-Free Water 10 mL RT U665516H Spin Columns RM with Collection Tubes 50 sets RT U665516I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTPreparation and important precautions before the experiment:1.To prevent RNase pollution, attention should be paid to the following aspects:1) RNase's plastic products and gun heads to avoid cross contamination.2) Prepare the solution using water without RNase.3) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction.3. If TRIzon Reagent is found to have precipitates before use, it can be dissolved in a water bath at 56 ℃ for a few minutes.Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Usage：1. Sample processing1a. Organization: 30-50 mg of tissue is thoroughly ground in liquid nitrogen and 1 mL of TRIzon Reagent is added, or 1 mL of TRIzon Reagent is added to the tissue sample and homogenized. Attention: The sample volume should not exceed 10% of the volume of TRIzon Reagent.2a. Single layer cell culture: Remove the culture medium and add an appropriate amount every 10 cm ² Add 1 mL of TRIzon Reagent.3a. Cell suspension: Collect cells by centrifugation. Add 1 mL of TRIzon Reagent to every 5 × 10 µ m cell.2. After adding TRIzon Reagent, repeatedly blow a few times to fully crack the sample. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Add 200 to every 1 mL of TRIzon Reagent µ LTRIzon PaI ™， Cover the tube tightly, vigorously shake for 15 seconds, and let it sit at room temperature for 2 minutes.4. Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 10 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer, and the upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Move the upper aqueous phase to a new RNase Free centrifuge tube (provided).5. Add an equal volume of 70% ethanol (prepared without RNase water) to the obtained aqueous solution, invert and mix well.6. Add all the solutions obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple batches. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Preparation of DNase I mixture: Take 52 µ LRNase Free Water, add 8 to it µ L 10 x Reaction Buffer and 20 µ L DNase I (1 U/ µ L) Mix well and prepare to a final volume of 80 µ The reaction solution of L.9. Directly add 80 µ L DNase I mixture to the adsorption column and incubate at 20-30 ℃ for 15 minutes.10. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.11. Add 500 to the adsorption column µ L Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.12. Repeat step 11.Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes and thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (enzyme digestion,. )PCR, etc.14. Place the adsorption column in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ L. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 14 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 14... Read More |