S. mutans that has been exposed to the compounds or control bacteria were washed in PBS and incubated with various fluorescent dyes according to the aim of the experiment before being analyzed on flow cytometer. A 20 min staining solution of 3.3 µM SYTO 9 together with 2 µg/mL PI was used to analyze bacteria with disrupted membrane [35 (link)]. This staining was performed at room temperature. For determining the membrane potential, the bacteria were exposed to 15 μM 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)) solution in PBS for 30 min at room temperature prior to flow cytometry [20 (link)]. For determining the membrane and DNA content, the bacteria were exposed to 10 µg/mL Nile red (APExBIO, Houston, TX, USA) and 1 µg/mL DAPI (Sigma) for 30 min at 37 °C [20 (link)]. The following excitation/emission parameters were used: 488 nm/531 nm for SYTO 9 and the green fluorescence of DiOC2(3); 488 nm/620 nm for PI and the red fluorescence of DiOC2(3); 561 nm/635 nm for Nile red and 355 nm/450 nm for DAPI. The samples were analyzed by the LSR-Fortessa flow cytometry instrument (BD Biosciences, San Jose, CA, USA), and 50,000 events were collected using the BD FACSDiva software 6.0. The FCS Express 7 software was used for analyzing the data.
Nile red
Manufactured by Apexbio
Sourced in United States
Nile Red is a fluorescent dye used for the detection and quantification of lipids and lipid-containing structures in biological samples. It is a lipophilic stain that selectively partitions into hydrophobic environments, such as lipid droplets, membranes, and other lipid-rich cellular components, resulting in a fluorescent signal that can be detected using appropriate microscopy or spectroscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software 6, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsr fortessa flow cytometry instrument, by BD (1 mentions)
Lrsfortessa flow cytometer, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Tcs sp8, by Leica (1 mentions)
Rhodamine 6g, by Merck Group (1 mentions)
Ethidium bromide etbr, by Merck Group (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
6 protocols using nile red
1
Flow Cytometric Analysis of S. mutans
2
Bacterial Membrane Staining and Analysis
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Control or AEA-treated bacteria were exposed to 1 µg/mL of diamidino-2-phenylindole (DAPI) for 30 min together with 10 µg/mL Nile Red (APExBIO, Boston, MA, USA) for 30 min at 37 °C. After incubation, the samples were analyzed by flow cytometry (LRS-Fortessa flow cytometer, BD Biosciences). The 355 nm laser was used for DAPI, and the fluorescence was collected by the blue (450 nm) filter. DAPI is a blue-fluorescent probe that fluoresces brightly upon selectively binding to DNA [11 (link)]. The Nile Red was analyzed using the 561 nm yellow–green laser excitation, and data were collected using the 635 nm filter [20 (link)].
3
Nile Red Membrane Staining
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Control or AEA-treated bacteria were exposed to 10 μg/ml nile red (APExBIO) for 30 min to stain the membranes64 (link). After washing the cells in PBS, the bacteria were analyzed on flow cytometry (LSR-Fortessa instrument, BD Biosciences) using the 561 nm yellow-green laser excitation and collecting the data using the 635 nm filter.
4
Biofilm Staining Using FITC and Nile Red
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Dimethyl sulfoxide (DMSO) was utilized to prepare storage solutions of 1 mg/mL and 2 mg/mL fluorescein isothiocyanate (FITC, Fujifilm, Wako, Japan) and Nile Red (APExBIO, Houston, TX, USA) separately. The hydrophobic filters were used to filter the dyes, and the solutions were stored in the dark at −4 °C. For FITC staining, 20 mg of biofilm sample was taken, and 1 mL of phosphate buffer solution (50 mM, pH = 9) was added, followed by the addition of 50 μL FITC. After dark incubation on a rotator for more than 8 h, the reaction was terminated by adding 5 M ammonium chloride. Subsequently, for Nile Red staining, 3 μL of 2 mg/mL Nile Red dye was added to the FITC-stained samples. After avoiding light exposure, staining was carried out for 5–10 min, and 10–20 μL was used for slide preparation. The samples were observed under excitation light at 488 nm and 552 nm. A confocal laser scanning microscope (CLSM, Leica TCS SP8, Wetzlar, Germany) was used to observe the biofilm activity, and 6–8 images were collected at random for each sample to avoid experimental error.
5
Assessing Bacterial Efflux Pump Activity
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To follow the intracellular dye levels in control and AA-treated bacteria, they were resuspended in 1 ml of PBS containing either of the following efflux pump substrates: 10 μg/ml Nile Red (APExBIO, Houston, TX, USA), 10 μM Rhodamine 6G (Sigma, St. Louis, MO, USA) or 2 μg/ml Ethidium bromide (EtBr, Sigma, St. Louis, MO, USA) for 30 min in PBS. The bacteria were then washed in PBS, and the fluorescence intensities were measured at various time points in a Fortezza flow cytometer using the excitation/emission of 561 nm/635 nm for Nile Red, and 488 nm/620 nm for Rhodamine 6G and Ethidium bromide (Banerjee et al., 2021 (link)). Nile Red emits red fluorescence when integrated into the membrane, but it is also a substrate for efflux pumps (Bohnert et al., 2010 (link)). Ethidium bromide which binds to both DNA and RNA, and Rhodamine 6B which has no known binding partners in bacteria, are fluorescent dyes that are commonly used in measuring efflux pump activities (Paixão et al., 2009 (link); Zeng et al., 2017 (link); Whittle et al., 2019 (link); Lu et al., 2020 (link); Banerjee et al., 2021 (link)).
6
Nile Red and DAPI Staining for Flow Cytometry
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Control bacteria (OD600nm = 0.3) or bacteria that have been exposed to CBG (0, 1.25, 2.5, 5, and 10 μg/ml) for 2 h, were stained with 10 μg/ml Nile red (APExBIO, Boston, MA, United States) and 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma) for 30 min at 37°C (Sugimoto et al., 2017 (link)). After washing the cells in PBS, the bacteria were analyzed on flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences) using the 561 nm yellow-green laser excitation and collecting the data using the 635 nm filter.
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