C6 NBD Ceramide (d18:1/6:0) (Synonyms: C6 NBD Ceramide, NBD Cer(18:1/2:0), NBD Ceramide (d18:1/6:0) , N-C6-NBD-D-erythro-Sphingosine)

This plan only provides a guide, please modify it to meet your specific needs.

1. Prepare dyeing solution

(1) Prepare stock solution: Take out the cryogenically stored C6 NBD Ceramide from the refrigerator and let it stand at room temperature to return to room temperature. Centrifuge at low speed to concentrate the powder at the bottom of the test tube. Add anhydrous DMSO to the tube to dissolve the powder and prepare a stock solution with a concentration of 5-10mM.

Note: Unused stock solution is aliquoted according to single dosage and stored in the dark at -20°C to avoid repeated freezing and thawing.

(2) Prepare working solution: Use HBSS/HEPES to dilute the stock solution at a ratio of 1:1000 to prepare a working solution with a concentration of 5-10μM.

Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it now.

2. Fluorescent labeling of Golgi apparatus in living cells

(1) Culture adherent cells on sterile coverslips;

(2) Remove the coverslip from the culture medium, aspirate the excess culture medium, remove the cell culture medium, and wash the cells growing on the coverslip with a suitable buffer (such as HBSS/HEPES);

(3) Add about 100 μL of dye working solution from one corner of the coverslip, shake gently to make the dye evenly cover all cells, and incubate at 4°C in the dark for 30 minutes;

Note: The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.

(4) Aspirate the staining solution, wash the cells 2-3 times with pre-cooled cell culture medium in an ice bath, replace with fresh culture medium and incubate at 37°C for another 30 minutes;

(5) Wash again with fresh culture medium, and then observe with a fluorescence microscope or confocal microscope. At this time, bright and strong fluorescent staining of the Golgi apparatus can be observed, while other membrane systems in the cell show relatively weak fluorescent staining.

3. Fluorescent labeling of Golgi apparatus in fixed cells

(1) Culture adherent cells on sterile coverslips;

(2) Wash the cells on the coverslip with HMEM. The cells were then fixed in glutaraldehyde or paraformaldehyde for 5-10 min (25°C);

(3) Use glutaraldehyde or paraformaldehyde to treat at room temperature for 5-10 minutes to fix the cells;

Note: Avoid using detergents and methanol/acetone fixatives to treat cells.

(4) Wash fixed cells in balanced salt solution (such as HCMF);

(5) Transfer the culture dish containing the coverslip to an ice-water bath and incubate with freshly prepared NaBH4 for 3-5 min (for glutaraldehyde-fixed cells);

(6) Use ice bath pre-cooled HCMF to wash the cells 2-3 times, each time for about 10 minutes;

(7) Return to room temperature, add about 100 μL of dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 60 minutes;

(8) Discard the staining solution, wash the fixed cells with HCMF, and incubate with 10% fetal calf serum or 2 mg/mL BSA (30-90min; 25°C (room temperature)) to back-exchange excess cells from the preparation solution. C6 NBD Ceramide, and enhances Golgi staining.

4. Microscope detection: The maximum absorption/emission wavelength of C6 NBD Ceramide is 466/536nm.

Precautions:

① C6 NBD Ceramide is suitable for staining fixed cells, while BODIPY FL C5-Ceramide (Cat. No.: GC68793) and BODIPYTR-ceramide are not suitable.

② Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching.

③ For your safety and health, please wear a lab coat and disposable gloves.

References:

[1].Pagano RE. A fluorescent derivative of ceramide: physical properties and use in studying the Golgi apparatus of animal cells. Methods Cell Biol. 1989;29:75-85.