| Description | Inquire | 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 | 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 | M666110 Component 96 T Storage M666110A Buffer WSL 40 mL RT M666110B Buffer MSL 40 mL RT M666110C Buffer CW1 (concentrate) 90 mL RT M666110D Buffer GW1 (concentrate) 40 mL RT M666110E Buffer GW2 (concentrate) 50 mL RT M666110F Buffer EB 30 mL RT M666110G Proteinase K 4×1.25 mL RT M666110H M666110 Component 96 T Storage M666110A Buffer WSL 40 mL RT M666110B Buffer MSL 40 mL RT M666110C Buffer CW1 (concentrate) 90 mL RT M666110D Buffer GW1 (concentrate) 40 mL RT M666110E Buffer GW2 (concentrate) 50 mL RT M666110F Buffer EB 30 mL RT M666110G Proteinase K 4×1.25 mL RT M666110H Magbeads V3 2×1 mL RTProduct Introduction：The reagent kit provides a simple, fast, and efficient method for extracting genomic DNA from blood samples. In the presence of high salt, DNA binds to the surface of silica coated Magheads. After rinsing, high-purity DNA is eluted in Buffer EB or deionized water. The purified DNA has good purity (A260/280 ratio between 1.7-1.9) and high integrity (>15 kb), and can be used for downstream experiments such as second-generation sequencing, quantitative PCR, and chip detection.Self provided instruments and reagents1) Constant temperature mixer2) 2/15 ml magnetic frame3) 32 channel nucleic acid extractor4) 96 channel nucleic acid extractor5) 96 DW Plate6) 8 channel Comb7) Spin tips pack8) Anhydrous ethanolPreparation and important precautions before the experiment1.Before the first use, add anhydrous ethanol to Buffer CW1, Buffer GW1, and Buffer GW2 according to the label of the reagent bottle and mark them properly.2.Magheads are strictly prohibited from freezing or centrifugation. Freezing and centrifugation may cause irreversible damage to Magheads.Operation stepsI. Manual single tube operation1. Use punching forceps to take 1 blood spot with a diameter of 6 mm or 4 blood spots with a diameter of 3 mm (depending on the actual situation) from the blood spot and place them in a 2.0 mL centrifuge tube.2. Add 40 to the centrifuge tube µ L Protein K and 300 µ L Buffer WSL, then place the centrifuge tube on a constant temperature mixer at 75 ℃ and 1200 rpm, shake and crack for 45 minutes to form Lysate. Remove the centrifuge tube from the constant temperature mixer, centrifuge briefly, and take the supernatant.Attention: If there is no constant temperature mixer, vortex the centrifuge tube for 10 seconds and incubate it in a 75 ℃ water bath for 30 minutes. During this period, vortex every 10 minutes for 10 seconds.3. Suck the supernatant into a new 2.0 mL centrifuge tube and add 300 µ L Buffer MSL, 300 µ L isopropanol and 20 µ L Magheads V3. Afterwards, place the centrifuge tube on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and crack for 15 minutes, or invert the centrifuge tube and mix continuously for 15 minutes.4. Place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, discard the solution thoroughly (keep the centrifuge tube fixed on the magnetic stand).5. Remove the centrifuge tube from the magnetic frame and add 900 µ L Buffer CW1 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm to shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).6. Remove the centrifuge tube from the magnetic frame and add 500 µ L Buffer GW1 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm to shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).7. Remove the centrifuge tube from the magnetic frame and add 900 µ L Buffer GW2 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, then place it on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).8. Remove the centrifuge tube from the magnetic frame and add 300 µ After shaking with 75% ethanol for 1 minute or 5 seconds, place the mixture on a constant temperature mixer at 25 ℃ and 1600 rpm for 2 minutes (ensure that the Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).9. Keep the centrifuge tube fixed on the magnetic frame, use a pipette to further remove the solution from the bottom and cover of the centrifuge tube, and then leave it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely.10. Remove the centrifuge tube from the magnetic frame and add 50-200 µ L Buffer EB. Vortex oscillation causes the magnetic beads to completely suspend in the eluent and then place them on a constant temperature mixer at 56 ℃ and 1600 rpm for 10 minutes of shaking and elution, or incubate the centrifuge tube in a 56 ℃ water bath for 10 minutes, with vortex oscillation every 3 minutes for 10 seconds.11. Place the centrifuge tube on a magnetic stand and let it stand for 2 minutes. After Magheads are completely adsorbed on the side wall of the centrifuge tube, transfer the eluent to a new centrifuge tube using a pipette and store at -20 ℃ for later use.II. Matching with CWE21001. Use punching forceps to take 1 blood spot with a diameter of 6 mm or 4 blood spots with a diameter of 3 mm (depending on the actual situation) from the blood spot and place them in a 2.0 mL centrifuge tube.2. Add 40 to the centrifuge tube µ L Protein K and 300 µ L Buffer WSL, then place the centrifuge tube on a constant temperature mixer at 75 ℃ and 1200 rpm, shake and crack for 45 minutes to form Lysate.3. Add the corresponding reagents to the 96DW deep well plate according to the table below. Position Reagent 1&7 Colume Lysate: All Buffer MSL: 300 µL isopropanol：300 µL Magbeads V3: 20 µL 2&8 Colume Buffer CW1: 900 µL 3&9 Colume Buffer GW1: 500 µL 4& 10 Colume Buffer GW2: 900 µL 5& 11 Colume 75%ethanol: 300 µL 6& 12 Colume Buffer EB: 70 µL4.Place the deep well plate and magnetic sleeve that have been added to the reagent at the corresponding positions of CWE2100/CWE3200, run the blood slide extraction program, and after about 40 minutes, the program ends. Remove the deep well plate and magnetic sleeve.5.Transfer the elution products from columns 6 and 12 of the deep well plate to a 1.5 mL centrifuge tube for low-temperature storage.III. Matching with CWE9601. Use punching forceps to take 1 blood spot with a diameter of 6 mm or 4 blood spots with a diameter of 3 mm (depending on the actual situation) from the blood spot and place them in a 2.0 mL centrifuge tube.2. Add 40 to the centrifuge tube µ L Protein K and 300 µ L Buffer WSL, then place the centrifuge tube on a constant temperature mixer at 75 ℃ and 1200 rpm, shake and crack for 45 minutes to form Lysate.3. Add the corresponding reagents to the 96DW deep well plate according to the table below Position Reagent Plate 1 Lysate: All Buffer MSL: 300 µL isopropanol ：300 µL Magbeads V3: 20 µL Plate 2 Buffer CW1: 900 µL Plate 3 Buffer GW1: 500 µL Plate 4 Buffer GW2: 900 µL Plate 5 75% ethanol : 300 µL Plate 6 Buffer EB: 70 µL4. Place the deep well plate and magnetic sleeve that have been added to the reagent at the corresponding positions on CWE960, run the blood slide extraction program, and after about 40 minutes, the program ends. Remove the deep well plate and magnetic sleeve.5. Transfer the elution products from Plate 6 to a 1.5 mL centrifuge tube for low-temperature storage... Read More | Inquire |