4-Di-16-ASP

This protocol provides a guide only and should be modified according to your specific needs.

1. Preparation of cell membrane staining solution

(1) Prepare DMSO or EtOH storage solution: the storage solution is prepared with DMSO or EtOH, the concentration is 1~5mM. For example: Dissolve 25mg of DiA (Mw: 787.04g/mol) in 6.35ml of anhydrous DMSO, and obtain a 5mM stock solution after fully dissolving.

Note: Store unused stock solutions in aliquots at -20°C, avoid repeated freezing and thawing.

(2) Preparation of working solution: Dilute the stock solution with a suitable buffer (such as: serum-free medium, HBSS or PBS) to prepare a working solution with a concentration of 0.5-5 μM.

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

2. Suspension Cell Staining

(1) The suspended cells were centrifuged at 4°C, 1000-1500rpm for 3-5 minutes, and the supernatant was discarded. Wash twice with PBS for 5 minutes each.

(2) Add 1 mL of DiA working solution to resuspend the cells and incubate at room temperature for 5-30 minutes.

Note: Different cells have different optimal culture time, 20min can be used as the initial incubation time, and then optimized to ensure uniform labeling results.

(3) After the incubation, centrifuge at 1000-1500rpm for 5 minutes, remove the supernatant, add PBS to wash 2-3 times, 5 minutes each time.

(4) Resuspend the cells with pre-warmed serum-free cell culture medium or PBS. Observed by fluorescence microscopy or flow cytometry.

3. Staining of Adhesive Cells

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, suck out the excess medium, and place the coverslip in a humid chamber.

(3) Add 100uL of dye working solution from one corner of the cover slip and shake gently to make the dye evenly cover all the cells.

(4) Incubate at room temperature for 5-30 minutes. Different cells have different optimal culture time, 20min can be used as the initial incubation time, and then optimized to ensure uniform labeling results.

(5) Discard the dye working solution after incubation, and wash the coverslip 2-3 times with pre-warmed culture solution.

4. Microscope detection: DiA (4-Di-16-ASP) has a very broad emission spectrum, which can be detected with green, orange and red filters. The maximum excitation/emission wavelength is 491/613nm. It is recommended to use XF21 filter from Omega Company Tablet and Chroma's 31024 were tested.

Note:

1) Fluorescent dyes have quenching problems, please try to avoid light to slow down fluorescence quenching.

2) For your safety and health, please wear a lab coat and disposable gloves for operation.