| 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 | 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 | DescriptionCobalt is a transition metal that serves as a trace dietary mineral for all multicellular organisms. Cobalt is an important cofactor for the Vitamin B12class of compounds where it occupies the center of the vitamin B12corrin ring. Cobalt can also be coordinated in the active site of the DescriptionCobalt is a transition metal that serves as a trace dietary mineral for all multicellular organisms. Cobalt is an important cofactor for the Vitamin B12class of compounds where it occupies the center of the vitamin B12corrin ring. Cobalt can also be coordinated in the active site of the non-corrin containing metalloenzyme methionine aminopeptidase.Suitability: Suitable for quantitating cobalt concentrations in a variety of samplesPrinciple: The Cobalt Assay kit provides a simple and direct procedure for measuring cobalt in a variety of samples. In this assay, cobalt reacts with 2-mercaptoethanol under basic conditions to form a complex with a strong absorbance at 475 nm. Interference from the metal ions Fe2+, Cu2+, Ni2+, Zn2+, and Mn2+is <10% at this wavelength. This assay gives a linear range of 10-50 nmoles of cobalt.}Preparation instructionsSuitable for quantitating cobalt concentrations in a variety of samplesPrincipleThe Cobalt Assay kit provides a simple and direct procedure for measuring cobalt in a variety of samples. In this assay, cobalt reacts with 2-mercaptoethanol under basic conditions to form a complex with a strong absorbance at 475 nm. Interference... Read More | Inquire | 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 |