Did (cell membrane red fluorescent probe)

​Did dye is a member of lipophilic fluorescent dye family, which can be used to dye cell membranes and other lipid soluble biological structures. When did is combined with the cell membrane, its fluorescence intensity is greatly enhanced. This kind of dye has a high quenching constant and excited state lifetime. Once the cell is stained, this kind of dye spreads on the whole cell membrane, and the whole cell membrane can be stained at the optimal concentration. Did (far red fluorescence) can be used for imaging and flow analysis of living cells. Did can be excited by 633 nm he – Ne laser. It has a longer excitation wavelength and emission wavelength than DiI (a common cellular fluorescent dye), which is more valuable in cell and tissstaining. The fixation of paraformaldehyde (methanol and other reagents are not allowed) can be carried out after did staining, but permeabilization after staining is not recommended. In addition, after fixed permeabilization (permeabilization with 0.1% TritonX-100 at room temperature), the plasma membrane staining can also be well performed. At 100 per use μ L of dyeing working solution with a concentration of 5 μ According to m calculation, 10 mg of working fluid can be used for 19009 times.Product parameters：Ex/Em (MeOH) = 644/663 nmMatters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when did is used to stain fixed cells or tissue samples, 4% paraformaldehyde prepared in PBS is usually used for fixation. The use of other inappropriate fixatives will lead to high fluorescence background. 3. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Cell membrane fluorescent dye ; anterograde and retrograde tracing of neurons ; long-term cell tracingUsage:1. Preparation of staining solution(1) Preparation of storage solution: The storage solution is prepared with anhydrous DMSO or EtOH, with a concentration of 1-5mM.Note: Unused storage liquids should be packaged and stored at -20 ° C to avoid repeated freeze-thaw cycles.(2) Preparation of working solution: Dilute the storage solution with a suitable buffer (such as serum-free medium, HBSS or PBS) and prepare a concentration of 1-5 μ M's working fluid.Note: It is recommended to optimize the final concentration of the working solution based on different cell lines and experimental systems. It is recommended to start exploring the optimal concentration within a range of 10 times the recommended concentration.2. Suspension cell staining(1) Add an appropriate volume of staining solution and resuspend the cells to a density of 1 × 106/mL.(2) Incubate cells at 37 ° C for 2-20 minutes, and the optimal culture time varies for different cells. 20 minutes can be used as the starting incubation time, and then the system can be optimized to obtain a uniform labeling effect.(3) After incubation, centrifuge at 1000-1500 rpm for 5 minutes. Pour the supernatant and slowly add the growth culture medium preheated at 37 ° C again to resuspend the cells.(4) Repeat step (3) more than twice.3. Staining of adherent cells(1) Cultivate adherent cells on sterile cover slips.(2) Remove the cover glass from the culture medium and absorb excess culture medium, but keep the surface moist.(3) Add 100 to one corner of the cover glass μ Gently shake the dye working solution of L to evenly cover all cells with the dye.(4) Incubate cells at 37 ° C for 2-20 minutes, and the optimal culture time varies for different cells. 20 minutes can be used as the starting incubation time, and then the system can be optimized to obtain a uniform labeling effect.(5) Dry the dye working solution, wash the cover glass with culture medium 2-3 times, cover all cells with preheated culture medium each time, incubate for 5-10 minutes, and then dry the culture medium. But keep the surface moist.4. Result detectionThe sample can be detected in the culture medium and analyzed by fluorescence microscopy imaging or flow cytometry. Molecular Formula: C67H103ClN2O3S. Molecular Weight: 1052.10.