7-Aminoactinomycin D (7-AAD) (Synonyms: 7-AAD, NSC 239759)

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare 7-Aminoactinomycin D (7-AAD) staining solution
(1) 7-Aminoactinomycin D (7-AAD) dye stock solution: Use 50% DMSO to dissolve 7-AAD into a 1mg/ml stock solution.
Note: Unused storage solution should be aliquoted and stored in the dark at -20°C to avoid repeated freezing and thawing. The prepared storage solution needs to be filtered and sterilized using a 0.22μm nylon filter.
(2) Preparation of working solution: Dilute the storage solution with a suitable sterile buffer (such as serum-free culture medium, HBSS or PBS) to prepare a working solution with a concentration of 0.5-20 μg/ml.
Notice:
① Low concentration (0.5-5μg/ml) 7-AAD is often used for flow cytometry staining or excluding dead cells; higher concentration (10-20μg/ml) 7-AAD is used to distinguish living cells (7-AAD negative ), apoptotic cells (7-AAD dim bright) and dead cells (7-AAD)
② Please adjust the concentration of the working fluid according to the actual situation and prepare it now.
2. Cell suspension staining
(1) Suspension cells: Centrifuge the suspended cells at 4°C and 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS for 5 minutes each time.
(2) Adherent cells: Discard the culture medium and add trypsin to digest the cells. After centrifugation and discarding the supernatant, add PBS and wash twice, 5 minutes each time.
(3) Resuspend approximately 1 x 106 cells in 0.5-1mL working solution and incubate at 4°C in the dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
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 100 μL of Hoechst 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 5-30 minutes.
(4) Aspirate away the dye working solution, wash the coverslip 2 to 3 times with culture solution, and observe with a fluorescence microscope.
4. Fluorescence detection: The maximum absorption wavelength of 7-AAD is 546 nm, and it can be effectively excited using a 543nm helium-neon laser; it can also be excited with a lower efficiency using a 488nm or 514nm argon laser line. The emission of 7-AAD has a very large Stokes shift, maximum in deep red: 647 nm. Therefore, 7-AAD is compatible with most blue and green fluorophores and even many red fluorophores in multicolor applications.

Precautions:
① 7-AAD is a strong carcinogen. In order to avoid skin and eye contact, it is recommended to wear gloves, lab coats and eye masks/face masks during operation;
② Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching.