DiI Cell Membrane Red Fluorescent Staining Kit, 100-1000 T

Product Information

Product Description/Introduction

Dil, also known as DilC18(3), 1,1`-Dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate, with a molecular weight of 933.87, is a class of lipophilic, long-chain dialkyldicarbocyanine dyes, fluorescent dyes, commonly used in the labeling of cell membranes and other fat-soluble biological structures. After entering the cell membrane, Dil can diffuse laterally and gradually stain the whole cell membrane.Dil fluorescence is very weak before entering the cell membrane, and the fluorescence intensity will be greatly enhanced when it binds to the cell, and it can emit orange-red fluorescence after excitation and can be detected by standard TRITC filters. The maximum excitation wavelength of Dil is 550 nm, and the maximum emission wavelength is 564 nm. According to the characteristics of Dil, it can stain living cells as well as fixed cells. In addition. Dil probes generally do not affect the viability of cells, so forward or reverse labeled cells or some substances (exosomes) can be used as tracer detection.

The Dil Cell Membrane Red Fluorescence Staining Kit contains Dil fluorescence probe and optimized staining buffer, which can make cell membrane staining faster, fluorescence more bright and stable.

Storage and Shipping Conditions

Ship with dry ice; store at -20°C in the dark, valid for 6 months.

Product Contents

Assay Protocol / Procedures

1. Preparation of Dil staining working solution:

1.1. Mix and dilute Dil Cell Membrane Red Fluorescent Probe with Staining Buffer at a ratio of 1:250 to prepare Dil Staining Working Solution (ready to use); note that the Dil probe dilution ratio can be adjusted at 1:250-1:500 according to the specific situation in order to obtain the best staining effect.

2. Staining of live suspension cells

2.1. Centrifuge the suspended cells at 500-1,000×g at room temperature for 3-5 min, remove the cell supernatant;

2.2. Add appropriate amount of Dil staining working solution to resuspend cells to a final cell density at 1-2×106 cells/mL.

2.3. Incubate at 37 in the dark for 5-20 min. The optimal incubation time varies for different cells. Start with 5 min and optimize the incubation time based on the final staining result.

2.4. At the end of incubation, centrifuge at 500-1000 g for 3-5 min at room temperature and aspirate the Dil staining working solution;

2.5. Cells were resuspended with pre-warmed PBS or cell culture medium at 37°C and centrifuged at 500-1000 g for 3-5 min to remove the supernatant;

2.6. Repeat step 2.5.

2.7. Detected by flow cytometry directly or by fluorescence microscopy after transfering cells to a multi-well plate, cell culture dish or cell climbing slide. Dil has an excitation maximum at 550 nm and an emission maximum at 564 nm.

3. Staining of live adherent live cells (6-well plate as an example):

3.1. Seed adherent cells in 6-well plate at a certain density.

3.2. Remove the culture medium and wash cells twice with PBS (Recommend G4202). Add 1 mL of Dil staining working solution (other size well plates, adjusted as appropriate to ensure that the dye covers the cells);

3.3. Incubate at 37 in the dark for 5-20min. The optimal incubation time varies for different cells. Start with 5 min and optimize the incubation time based on the final staining result.

3.4. Aspirate the cell membrane staining working solution and wash cells 1-2 times with preheated PBS or cell medium.

3.5. Add pre-warmed cell culture medium or cell medium at 37 and detect cells by fluorescence microscopy. Dil has an excitation maximum at 550 nm and an emission maximum at 564 nm.

4. Staining of fixed adherent cells:

4.1. Sample preprocessing:

For cells: Remove the cell medium and wash 1-2 times with PBS. Add 4% paraformaldehyde fix solution (Recommend G1101) for 10 min at room temperature. Remove fix solution and wash 2-3 times with PBS;

4.2. Permeabilization: Add 0.1-0.5% Triton-100 (prepared with PBS) and permeated for 10 min at room temperature. Remove permeabilization solution and wash 2-3 times with PBS.

4.3. (Optional, immunofluorescent labeling) Incubate with antibodies according to immunostaining protocol or incubate with other dyes.

Note: Blocking solution, antibody diluent, and wash solution for immunostaining should not contain detergents.

4.4. Add an appropriate amount of Dil staining working solution to cover the cells, incubate at 37°C away from light for 5-20 min, and aspirate the Dil staining working solution. It is recommended that the staining time be adjusted to the specific cell sample to obtain optimal staining results;

4.5. Cells were washed 2-3 times with PBS and then placed under a fluorescence microscope for observation (cells need to be covered with appropriate amount of PBS).Dil has a maximum excitation wavelength of 550 nm and a maximum emission wavelength of 564 nm.

5. Fluorescent labeling of exosomes:

5.1. Resuspended exosome precipitation with an appropriate amount of Dil staining working solution.

5.2. Incubate at 37 for 30 min in the dark.

5.3. (optional) Dilute the sample with 10-fold volume PBS.

5.4. Extracted exosomes again according to the previous extraction protocol to remove excess dyes.

5.5. Collect exosomes precipitation and resuspended in PBS to obtain Dil-labeled exosomes. It can be used for subsequent experiments, such as cellular uptake.

Note

1. All fluorescent dyes have quenching problems, please protect from light during the operation to slow down fluorescence quenching.

2. Due to the probe is lipophilic, please avoid using reagents containing glycerin or other organic matter.

3.  If fixation is required, we recommend to fix in 4% paraformaldehyde. Other inappropriate fix solutions will lead to high fluorescence background.

4. The optimum dilution ratio and incubation time of the probe should be adjusted according to the actual situation due to the different sensitivity between cells and experimental requirement.

5. For your safty and health, please wear safety glasses, gloves, or protective clothing.

For Research Use Only!

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