Nonyl acridine orange

Nonyl acridine orange [3,6-bis(Dimethylamino)-10-nonyl-acridinium bromide] (NAO) and FM4-64 were purchase from Molecular Probes. 1,2-Dioleoyl-sn-glycero-3-phosphoethano lamine-N-(lissamine rhodamine B sulfonyl) (DOPE-rho), 1,2-myristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (DMPE-NBD); 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phos pho-glycerol (NBD-PG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) were purchased from Avanti polar lipids. NBD-labeled lipid II was a generous gift from E. Breukink (van Dam et al., 2007 (link)).

Mitochondrial mass was determined using nonyl acridine orange (NAO; λ_ex = 490 nm, λ_em = 518 nm; Invitrogen), which binds to cardiolipin independently of the energetic status of mitochondria (Benel et al. 1989 (link)). Exponentially growing cells were incubated in the dark with 1 µg/mL NAO for 20 minutes at 30°, followed by washing, resuspension in PBS, and flow cytometry of 100,000 cells (Lai et al. 2002 (link)).

The cells were washed with HEPES and stained with Nonyl Acridine Orange (NAO; 0.1 mM; Invitrogen) for 30 min at 37 °C in a 5% CO₂ incubator. The stained cells were washed twice with HEPES, and fluorescence was detected on an EVOS M5000 (Thermo Fisher).

Mitochondrial mass was measured by staining live cells with MitoTracker Red CMXRos (Thermo Fisher Scientifific, MA, USA) and Nonyl Acridine Orange (NAO) (Invitrogen, Carlsbad, CA). The nuclei were labeled with 4’, 6-diamidino − 2-phenylindole (DAPI, Beyotime, Shanghai, China). Mitochondrial fluorescence intensity was observed and photographed by fluorescence microscopy (Echo Laboratories, New York, USA).
Mitochondrial membrane potential (MMP) was estimated by JC-1 (Beyotime, Nanjing, China). Cells were incubated with 5 µM JC-1 for 30 min in the dark then washed and resuspended. The JC-1 aggregates or monomers were detected by immunofluorescence microscopy. JC-1 selectively enters into mitochondria where it can exist in two forms, monomeric or aggregate, depending upon the MMP. The green-shifted monomers tend to predominate under conditions with low MMP, whereas the red-shifted aggregates are favored under conditions with high MMP [19 (link)].

The WT strain, the mar1Δ mutant strain, and the mar1Δ + MAR1 complemented strain were incubated in YPD medium at 30°C and TC medium at 37°C for 18 h. Approximately 10⁷ cells from each sample were stained with 250 nM nonyl acridine orange (NAO) (Invitrogen) for 30 min at 30°C, 150 rpm shaking. All samples were then washed three times with 1X PBS and imaged by epifluorescence microscopy using a Zeiss Axio Imager A1 microscope equipped with an Axio‐Cam MRm digital camera. Cells were imaged by DIC and with a GFP filter to capture NAO staining. Identical exposure times were used to image all cells. Fiji software was used to process images. Mitochondrial morphology was assigned to cells using previously published criteria (Chang and Doering, 2018 (link)). Statistically significant differences in the percentages of cells within each strain with diffuse morphology were analyzed by one-way analysis of variance (ANOVA) and the Tukey-Kramer test (GraphPad Software, San Diego, CA).

The WT strain, the mar1Δ mutant strain, and the mar1Δ + MAR1 complemented strain were incubated in YPD medium at 30°C and TC medium at 37°C for 18 h. Approximately 10⁷ cells from each sample were co-stained with 250 nM nonyl acridine orange (NAO) (Invitrogen) and 100 nM MitoTracker^TM Red CMXRos (MT) (Life Technologies) for 30 min at 30°C, 150 rpm shaking. Cells were concurrently stained with 10 μM Sytox^TM Blue (SB) (Invitrogen) for 15 min at 30°C, 150 rpm shaking. Unstained cells were prepared alongside of stained samples. All samples were then washed three times with 1X PBS. Approximately 10⁶ cells from each sample were submitted to the Duke Cancer Institute Flow Cytometry Shared Resource for analysis on a BD Fortessa X-20 flow cytometer. NAO was excited using a 488 nm laser, MT was excited using a 516 nm laser, and SB was excited using a 405 nm laser. Data were analyzed using FlowJo v10.8.0 software (FlowJo, LLC). Relevant events were gated for live cells (FSC vs. SSC and SSC vs. SB), single cells (FSC-H vs. FSC-A), and positive labelling (SSC vs. NAO/MT, determined using unstained cells as negative controls).