| Product Description | The efficiency of DiO Plus is equivalent to Neuro-DiO. DiO Plus is a new type of dye that replaces the membrane green fluorescent dye DiO.Due to its low solubility, easy formation of polymers and slow lateral diffusion, DiO is difficult to be used in the study of neurons and cell suspensions. DiO Plus and DiO have almost the same absorption and emission spectra, but the former has better membrane solubility and does not form non-fluorescent aggregates, which usually slows down the diffusion rate of dyes on the membrane. The fixation of paraformaldehyde ( other reagents such as methanol cannot be used ) can be carried out after dyeing with DiO Plus, but the process of dialysis after dyeing is not recommended. In addition, plasma membrane staining can also be performed well after fixed permeabilization ( permeabilization with 0.1 % TritonX-100 at room temperature ). Based on the use of 100 μL dyeing working fluid at a time, the concentration of the dyeing working fluid is 3 μM, and 10 mg is configured as the working fluid, which can be used about 15,346 times. Product parameters: Ex/Em (MeOH) = 484/501 nm
Scope of application： Cell membrane fluorescent dye ; anterograde and retrograde tracing of neurons ; long-term cell tracing Usage:
1. Preparation of staining solution
(1) Configure DMSO or EtOH storage solution: Use DMSO or EtOH to prepare the storage solution with a concentration of 1-5 mM.
Note: Unused storage liquids should be packaged and stored at -20 ℃ 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 ℃ for 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 uniform labeling results.
(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, 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 uniform labeling results.
(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 detection
The sample can be detected in the culture medium and analyzed by fluorescence microscopy imaging or flow cytometry. Notes:
1. Before use, please centrifuge the product instantaneously to the bottom of the tube before conducting subsequent experiments.
2.When staining fixed cell or tissue samples with DiO Plus, 4% paraformaldehyde prepared in PBS is usually used for fixation. The use of other inappropriate fixation solutions can result in a high fluorescence background.
3. Fluorescent dyes all have quenching problems, please try to avoid light as much as possible to slow down fluorescence quenching.
4.For your safety and health, please wear laboratory clothes and disposable gloves when operating. |
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L2325209
