Acridine Orange 10-Nonyl Bromide (NAO) is a highly specific probe for cardiolipin and is often used to label CL-rich mitochondria. The maximum acridine orange excitation emission light is 489/525nm.
Acridine Orange 10-Nonyl Bromide (NAO), an acridine orange derivative, is a highly specific probe for cardiolipin (CL) and is commonly used to label CL-rich mitochondria[1]. Acridine Orange 10-Nonyl Bromide has been proven to specifically bind CL in vitro and is often used as a live cell stain to measure CL content[2]. In rat cortical astrocytes, neonatal cardiomyocytes, and isolated brain mitochondria, Acridine Orange 10-Nonyl binding to mitochondria was abolished by the mitochondrial uncoupler FCCP, demonstrating labeling of mitochondria by Acridine Orange 10-Nonyl Bromide Staining is highly dependent on membrane potential[2]. Acridine Orange 10-Nonyl has a maximum excitation/emission light of 489/525nm and has been used to quantify cardiolipin content and mitochondrial mass in isolated mitochondria and cellular systems[3].
References:
[1].Myriam E Rodriguez,et.Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization. 2008 Jun;8(3):237-46. doi: 10.1016 /j.mito.2008.04.003. Epub 2008 Apr 25.
[2].Jake Jacobson, Michael R Duchen, Simon J R Heales. Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass. 2002 Jul;82(2):224- 33. doi: 10.1046/j.1471-4159.2002.00945.x.
[3]. 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.
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.
