产品介绍

Product Information

Dilinoleyl Dil

Catalog Number: D4053

Product Size: 5 mg

Application Scope: Cell membrane fluorescent dyes, anterograde and retrograde tracing of neurons, long-term tracing of cells

ParametersAppearance: Dark red solid soluble in EtOH, DMSO or DMF Ex/Em (MeOH) = 549/565 nm

Molecular Formula: C59H89ClN2O4Molecular Weight: 925.8

Molecular Structure:

Storage

Store at 4℃ and protect from light. Expiration date marked on the outer packing.

Description

Dilinoleyl DiI, also known as FAST DiI, has identical absorption and emission spectra as DiI. Because Dlinoleyl DiI contains unsaturated hydrocarbon chains, the lateral diffusion rate of Dilinoleyl DiI on the cell membrane is faster than that of DiI. It is because of this characteristic that it is widely used in the tracking of neuronal tissue.

After Dilinoleyl DiI staining, cells can be fixed with paraformaldehyde (no other reagents such as methanol can be used), but it is not recommended to perform permeabilization after staining. In addition, after fixation and permeabilization (permeabilization with 0.1% TritonX-100 at room temperature), the plasma membrane can also be stained well.

Calculated by using 100 μL of staining working solution each time, and the concentration of staining working solution is 10 μM, 5 mg of working solution can be used about 5400 times.

Protocol

1. Dyeing liquid preparation

(1) Stock solution: Use DMSO, EtOH to make a stock solution with a concentration of 1 to 10 mM.

Note: It is recommended to store the storage solution at -20℃, and aliquot it in small quantities 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) to prepare a working solution with a concentration of 1 to 10 μM.

Note: The final concentration of the working solution is recommended to be optimized according to different cell lines and experimental systems. It is recommended to start the exploration of the optimal concentration from the range of 10 times the recommended concentration.

2. Suspension cell staining

(1) Suspend cells at a density of 1×106/mL in working solution.

(2) Incubate for 20 minutes at 37°C. The optimal incubation time will vary depending on cell type. Start with 20 minutes and optimize as needed for uniform labeling.

(3) Pellet the cells by centrifugation at 1000～1500 rpm for 5 minutes. Remove the supernatant and wash the cells by gently resuspending them in warm (37°C) medium.

(4) Repeat step (3) two more times

3. Adherent cell staining

(1) Adherent cells were grown on sterile coverslips.

(2) Remove the coverslip from the medium and aspirate the excess medium but keep the surface wet.

(3) Add 100 μL of the dye working solution to one corner of the coverslip and shake gently to cover all cells evenly with the dye

(4) Incubate cells at 37°C for 5-20 min, and the optimal incubation time varies for different cells. 20 min can be used as the initial incubation time, and then the system can be optimized to obtain uniform labeling effect.

(5) Aspirate the dye working solution dry, wash the coverslips 2~3 times with culture medium, cover all cells with pre-warmed medium each time, incubate for 5~10 min, and then blot dry the medium. but keep the surface moist.

4. Result detection

Samples can be assayed in culture and analyzed by fluorescence microscopy imaging or flow cytometry

Notes

1. Please centrifuge the product instantaneously to the bottom of the tube before use for subsequent experiments.

2. When using Dilinoleyl DiI to stain fixed cells or tissue samples, 4% paraformaldehyde in PBS is usually used for fixation. Using other inappropriate fixatives will lead to high fluorescence background.

3. There are quenching problems with fluorescent dyes. Please avoid light to slow down the fluorescence quenching.

4. For your safety and health, please wear lab coats and disposable gloves.