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

This protocol is only a guide. Please modify it according to your specific needs.
1. Prepare the staining solution
(1) Prepare the storage solution: Thaw the frozen product at room temperature and concentrate the powder at the bottom by brief centrifugation. Dissolve DAF-FM DA (Diaminofluorescein-FM diacetate) in anhydrous DMSO to prepare a storage solution with a concentration of 1-10mM.
Note: After the unused storage solution is aliquoted, store it in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Dilute the storage solution with a suitable buffer (e.g., serum-free and phenol red-free culture medium or PBS) to prepare a working solution with a concentration of 1-10μM.
Note:
① Please adjust the working solution concentration according to the actual situation and prepare it before use.
② Phenol red will interfere with the fluorescent signal generated by the dye. Please use a culture medium without phenol red to prepare the dye working solution.
2. Cell suspension staining (for example, six-well plate)
(1) Suspended cells: Centrifuge at 4°C, 1000g for 3-5 minutes, 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 3-5min after digestion.
(3) Add DAF-FM DA (Diaminofluorescein-FM diacetate) working solution to resuspend the cells and incubate at 37°C in the dark for 0.5-1h. The optimal incubation time varies for different cells. Please explore it yourself according to the specific experimental needs.
(4) After incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and wash 2-3 times with PBS, each time for 5 minutes.
(5) Resuspend the cells in pre-warmed serum-free cell culture medium or PBS, and observe by fluorescence microscopy or flow cytometry.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, aspirate 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 gently shake to evenly cover all cells with the dye.
(4) Incubate at 37°C in the dark for 0.5-1h. The optimal incubation time varies for different cells. Please explore according to the specific experimental needs.
(5) After the incubation, aspirate the dye working solution and wash the coverslip 2-3 times with pre-warmed culture medium.
4. Microscope detection: The maximum excitation/emission wavelength of DAF-FM DA (Diaminofluorescein-FM diacetate) is 495/515nm.
Note:
1) Fluorescent dyes all 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 when operating.
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.