DIA (cell membrane green fluorescent probe)

​Dia is a green fluorescent dye of cell membrane. Its diffusion speed in cell membrane is faster than DIO, and it is often used with DiI for dual color labeling of cell membrane. After dia staining, paraformaldehyde (methanol and other reagents cannot be used) can be fixed, but permeabilization process 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. The staining effect of dia on fixed cells was better than that of DIO. At 100 per use μ L of dyeing working solution with a concentration of 10 μ According to the calculation of M, 50 mg of working fluid can be used 63532 times.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when dia 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. 2. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 3. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Cell membrane fluorescent dye；mainly used for cell imaging, cell tracking and tracking.Parameters:Ex/Em (MeOH) = 491/613 nm  Usage:1. Preparation of staining solution(1) Preparation of storage solution: The storage solution is prepared with anhydrous DMSO, anhydrous DMF, or EtOH at a concentration of 1-5 mM. The solubility of DiA in anhydrous DMSO and anhydrous DMF is higher than that in EtOH.Note: a. Unused storage liquids should be packaged and stored at -20 ℃ to avoid repeated freeze-thaw cycles;b. When it is difficult to dissolve, it can be heated appropriately and treated with ultrasound to promote dissolution.(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 1-30 μ M's working fluid. The most commonly used working fluid concentration is 5-10 μ M.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 ℃ 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 ℃ 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 ℃ 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: C46H79IN2. Molecular Weight: 787.00. PubChem CID: 6439243. Isomeric SMILES: CCCCCCCCCCCCCCCCN(CCCCCCCCCCCCCCCC)C1=CC=C(C=C1)/C=C/C2=CC=[N+](C=C2)C.[I-].