DAF-FM DA (Diaminofluorescein-FM diacetate) (Synonyms: 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate, DAF-FM DA)

This plan only provides a guide, please modify it to meet your specific needs.
1. Preparation of staining solution
(1) Prepare stock solution: Place the frozen product at room temperature to thaw, and briefly centrifuge to concentrate the powder at the bottom. This product is 50μg per tube. Use anhydrous DMSO to dissolve DAF-FM DA and prepare a stock solution with a concentration of 1-5mM.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Use an appropriate buffer (such as serum- and phenol red-free medium or PBS) to dilute the stock solution and prepare a working solution with a concentration of 1-10μM.
Note:
① Please adjust the concentration of the working fluid according to the actual situation and prepare it for use.
② Phenol red will interfere with the fluorescence signal generated from the dye. Please use a medium without phenol red to prepare the dye working solution.
2. Cell suspension staining (using a six well plate as an example)
(1) Suspension cells: Centrifuge at 1000g for 3-5 minutes at 4°C, discard the supernatant, and wash twice with PBS for 5 minutes each time.
(2) Adherent cells: Wash twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.
(3) Add DAF-FM DA working solution to resuspend the cells, and incubate at 37 °C in the dark for 0.5-1h. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.
(5) Resuspend cells in pre-warmed serum-free cell culture medium or PBS and observe by fluorescence microscope or flow cytometry.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.
(4) Incubate at 37 °C in the dark for 0.5-1h. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(5) After the incubation, discard the dye working solution and use pre-warmed culture solution to wash the coverslip 2 to 3 times.
4. Microscope detection: The maximum excitation/emission wavelength of DAF-FM DA is 495/515nm.
Precautions:
1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves.

References:
[1]. Chris D St Laurent,Tae Chul Moon,A Dean Befus. Measurement of nitric oxide in mast cells with the fluorescent indicator DAF-FM diacetate. 2015:1220:339-45. doi: 10.1007/978-1-4939-1568-2_21.