Acridine Orange 10-Nonyl Bromide (Nonylacridine orange) (Synonyms: 10-nonyl Acridine Orange)

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

1. Prepare Acridine Orange 10-Nonyl Bromide staining solution

(1) Dye stock solution: Use DMSO to dissolve Acridine Orange 10-Nonyl Bromide（NAO） into a 1-5mM stock solution.

Note: It is recommended that unused stock solutions be aliquoted and stored in the dark at -4°C or -20°C (stored under nitrogen), and avoid repeated freezing and thawing.

(2) Dye working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare a dye working solution with a concentration of 20nM~1μM.

Note:

① Since the usage concentration of （NAO） is relatively low, it is recommended to use DMSO to dilute and prepare an intermediate storage concentration (for example: 100uM);

②  Please adjust and optimize the working fluid concentration according to the actual situation, and prepare it now.

2. Cell suspension staining(Take a 6-well plate as an example)

(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.

(2) Wash the adherent cells twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.

(3) Add 1 mL of dye working solution preheated at 37 °C to resuspend the cells, and incubate at 37 °C in the dark for 15-30 minutes. The optimal culture time for different cells is different.

(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS for washing 2-3 times.

(5) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through 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 100uL dye working solution from one corner of the coverslip, shake gently to make the dye evenly cover all cells, and incubate at 37 °C in the dark for 15-30 minutes.

(4) Aspirate and discard the dye working solution, use culture medium to wash the coverslip 2 to 3 times, and observe with a fluorescence microscope.

4. Microscope detection: The maximum excitation/emission light of Acridine Orange 10-Nonyl Bromide is 489/525nm.

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]. M H Ratinaud, P Leprat, R Julien. In situ flow cytometric analysis of nonyl acridine orange-stained mitochondria from splenocytes. 1988 May;9(3):206-12. doi: 10.1002/cyto.990090304.