Syto9

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, China, Germany, France, India, Japan, Australia

SYTO9 is a nucleic acid stain used for fluorescent labeling of DNA and RNA in living cells. It binds to both double-stranded and single-stranded nucleic acids, emitting green fluorescence upon binding.

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Lab products found in correlation

Propidium iodide, by Thermo Fisher Scientific (119 mentions) Alexa fluor 647, by Thermo Fisher Scientific (14 mentions) Propidium iodide, by Merck Group (12 mentions) Lsm 700, by Zeiss (12 mentions) Calcofluor white, by Merck Group (10 mentions) Alexa fluor 647 dextran conjugate, by Thermo Fisher Scientific (10 mentions) Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (9 mentions) Lsm 800, by Zeiss (9 mentions) Alexa fluor 647 labeled dextran conjugate, by Thermo Fisher Scientific (9 mentions) Lsm 710, by Zeiss (9 mentions) Mx3005p rt pcr instrument, by Agilent Technologies (8 mentions) Syto9, by Agilent Technologies (8 mentions) Lsm 510, by Zeiss (8 mentions) Syto9, by Leica camera (7 mentions) Syto 9, by Olympus (7 mentions) Sypro red, by Thermo Fisher Scientific (6 mentions) Thin walled pcr plate, by Agilent Technologies (6 mentions) Dimethyl sulfoxide (dmso), by Merck Group (6 mentions) Tcs sp5, by Leica camera (6 mentions) Fv1000, by Olympus (5 mentions)

524 protocols using syto9

Fluorescent Staining of Mammalian Cells and Biofilms

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Mammalian cells were incubated for 20 min in a 10 µM solution of CellTracker Orange CMRA (Invitrogen, C34551) and washed with DPBS before seeding the bacteria. For standard visualization of biofilm grown on top of epithelial monolayers, since V. cholerae strains were not constitutively fluorescent, samples were incubated for 20 min with a 10 µM solution of SYTO9 (Invitrogen, S34854) and washed with DPBS before visualization. This results in double staining of epithelial cells. For the visualization of live and dead cells in infected monolayers we instead incubated the samples for 20 min in a solution containing 5 µg/ml Hoechst (Thermo Fischer Scientific, 62249) and 5 µM Calcein-AM (Sigma Aldrich, 17783). For the visualization of epithelial cells monolayers permeability, we added 1 ml of a 2 µM solution of fluorescein isothiocyanate-dextran (Sigma Aldrich, 46944) on top of the cells and imaged after 30 min.
V. cholerae biofilms grown in microfluidic channels were incubated for 20 min with a 10 µM solution of SYTO9 (Invitrogen, S34854) and washed with M9 minimal medium before visualization.

Cont A., Rossy T., Al-Mayyah Z, & Persat A. (2020). Biofilms deform soft surfaces and disrupt epithelia. eLife, 9, e56533.

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Biofilm Quantification via CLSM

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Bacterial cells with an initial concentration of OD600 = 0.01 in LB broth medium were transferred into glass-bottomed dishes (Cellvis, D29-14-1-N) and incubated at 25°C for 24 or 48 h to form biofilms, respectively. Subsequently, supernatant was removed and the biofilms attached on the dish were rinsed gently three times in PBS. The biofilms were then stained with SYTO 9 (Invitrogen, L13152) according to the manufacturer’s instructions, briefly, 3 μM SYTO 9 stain was added to the biofilms and incubate at room temperature in the dark for 15 min. Then it was examined using confocal laser scanning microscopy (ZEISS LSM 880) with an excitation of 488 nm. Three-dimensional images of the biofilms were generated through Z-stack imaging, utilized the Zen Black software (Zeiss, Oberkochen, Germany). Image J was subsequently used to calculate the integrated density.

Liu H., Ma J., Yang P., Geng F., Li X., Lü J, & Wang Y. (2024). Comparative analysis of biofilm characterization of probiotic Escherichia coli. Frontiers in Microbiology, 15, 1365562.

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Confocal Microscopy for Biofilm Analysis

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Following incubation, biofilms were rinsed with 150 mM NaCl and refilled with TSB or BHI containing 5 μM Syto 9 (1:1000 dilution from a Syto 9 stock solution at 5 mM in DMSO; Invitrogen, France), a cell-permeable green fluorescent nucleic acid marker. The plate was then incubated in the dark at 30°C for 20 min to enable fluorescent labeling. Images were acquired using a Leica SP2 AOBS confocal laser scanning microscope (Leica Microsystems, France) at the MIMA2 microscopy platform¹. The excitation laser wavelength used for Syto 9 was 488 nm, and emitted fluorescence was recorded within the range 500–600 nm. Images (512 × 512 pixels) were acquired through a 63 × Leica oil immersion objective (numerical aperture, 1.4) with a z step of 1 μm and a frequency of 400 Hz. 3D projections were generated with the Easy 3D IMARIS function (Bitplane, Zurich, Switzerland). Biofilms structural parameters [biovolume (μm³), mean thickness (μm), and roughness (μm)] were extracted from image series using the PHLIP Matlab routine². Each value presented is the average of six image series acquired in three independent experiments.

Dubois-Brissonnet F., Trotier E, & Briandet R. (2016). The Biofilm Lifestyle Involves an Increase in Bacterial Membrane Saturated Fatty Acids. Frontiers in Microbiology, 7, 1673.

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Biofilm Imaging on Microtiter Plates

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Submerged biofilms were grown on the surface of polystyrene 96-well microtiter plates with a µclear^® base (Greiner Bio-one, France) enabling high-resolution fluorescence imaging as previously described [44 (link)]. An amount of 200 µL of an overnight culture in TSB (adjusted to an OD 600 nm of 0.02) was added in each well. The microtiter plate was then incubated at 30 °C for 90 min to allow the bacteria to adhere to the bottom of the wells. Wells were then rinsed with TSB to eliminate non-adherent bacteria and refilled with 200 µL of sterile TSB. The plates were incubated at 30 °C for 24 h, and 5 μM of the cell permeant nucleic acid dye SYTO 9 (diluted 1:1000 in TSB from a SYTO 9 stock solution at 5 mM in DMSO; Invitrogen, France) were added to the 200 µL culture, obtain green fluorescent bacteria. For each strain, at least 9 to 15 wells were analyzed independently.

Dergham Y., Sanchez-Vizuete P., Le Coq D., Deschamps J., Bridier A., Hamze K, & Briandet R. (2021). Comparison of the Genetic Features Involved in Bacillus subtilis Biofilm Formation Using Multi-Culturing Approaches. Microorganisms, 9(3), 633.

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Bacterial Biofilm Quantification via CLSM

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SYTO-9 and PI stains (Invitrogen Eugen, Oregon, USA) were used to stain live and dead bacteria, respectively, using established protocols.^{57 (link)} Briefly, 1.5 μl of a 30 mM PI concentration and 1.5 μl of 3.34 mM SYTO-9 were inserted into 1 ml of 100 mM NaCl. The sample membrane was then covered with the staining mix by pipetting, incubated for 10 min in the dark and gently washed three times with 100 mM NaCl. Two independent biofilm growth experiments were carried out for each of the cellulose variants. The developed biofilms were then visualized using a CLSM (Zeiss-Meta 510, Zeiss, Oberkochen, Germany), with images collected from eight positions on each membrane (representative images are shown in Supplementary Figs. S2–S4). Image processing and determination of specific biovolume values (μm³/μm²) were conducted using IMARIS 3D software (Bitplane, Zurich, Switzerland).

Ziemba C., Shabtai Y., Piatkovsky M, & Herzberg M. (2016). Cellulose effects on morphology and elasticity of Vibrio fischeri biofilms. NPJ Biofilms and Microbiomes, 2, 1.

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Quantifying Internalization and Viability of Neisseria gonorrhoeae in Neutrophils

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Baclight viability dyes (Life Technologies) were used to stain membrane permeant bacteria (propidium iodide) and total bacteria (Syto9) and analyzed as previously described (Johnson et al., 2013a ). Briefly, acid-washed glass coverslips were coated with 50% normal human serum at 37°C for 30 min prior to infection. Mid-log phase Gc were exposed to neutrophils for 1 hr at 37°C at an MOI of 1. The percent of Gc positive for propidium iodide was calculated relative to total bacteria (propidium iodide-positive and –negative) for the intracellular and extracellular compartments. From these experiments, percent internalization was also determined by dividing total intracellular bacteria by total bacteria, regardless of propidium iodide fluorescence. For experiments assessing serum opsonization and internalization, extracellular bacteria were stained with Alexa Fluor 647-coupled soybean lectin (ThermoFisher), and total bacteria were stained with 5 µM Syto9 (ThermoFisher) following neutrophil permeabilization; percent internalization was determined by dividing the number of intracellular bacteria by the number of cell-associated (internal and external) bacteria. At least 60 bacterial cells were assessed per strain for each independent experiment.

Ragland S.A., Schaub R.E., Hackett K.T., Dillard J.P, & Criss A.K. (2016). Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils. Cellular microbiology, 19(3), 10.1111/cmi.12662.

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Assessing Cell Viability in M. florum

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Cell viability of M. florum was assessed by SYTO 9 and PI double staining (Boulos et al, 1999). M. florum cells were centrifuged at 10°C for 2 min at 21,100 × g, and washed once with cold PBS1×. Cells were centrifuged again and then resuspended in PBS1× containing 5 µM SYTO 9 (Molecular Probes) and 10 µg/ml PI (Biotium). Cells were stained at RT for ~ 20 min. A fixed‐cells control was also performed by incubating a M. florum washed cell aliquot with 1% (w/v) formaldehyde at RT for ~ 25 min. Fixed cells were centrifuged at 10°C for 2 min at 21,100 × g, resuspended in PBS1× containing 0.1% (v/v) Triton X‐100, and incubated at RT for 2 min. Cells were centrifuged again and finally resuspended in PBS1× containing 5 µM SYTO 9 (Thermo Fisher Scientific) and 10 µg/ml PI (Biotium). Samples were immobilized on agarose pad slides and examined by widefield fluorescence microscopy using an Axio Observer Z1 inverted microscope (Zeiss) equipped with an AxioCam 506 mono (Zeiss) camera and a 100×/NA1.4 Plan‐Apochromat oil immersion objective. SYTO 9 and PI were excited and acquisitioned using GFP and Cy3 excitation/emission filters, respectively. Images were captured with Zeiss Zen 2.0 imaging software and analyzed using Fiji (Schindelin et al, 2012).

Matteau D., Lachance J., Grenier F., Gauthier S., Daubenspeck J.M., Dybvig K., Garneau D., Knight T.F., Jacques P, & Rodrigue S. (2020). Integrative characterization of the near‐minimal bacterium Mesoplasma florum. Molecular Systems Biology, 16(12), e9844.

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Propidium Iodide and Syto9 Fluorescence Imaging

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For propidium iodide (PI)/Syto9 imaging, 20 μL of 100 μg/mL PI (Thermo Fisher) and 20 μL of 100 μM Syto9 (Thermo Fisher) were added to a 20-mL universal flask along with 2 mL of exponential phase culture, which had been incubated for 3.5 h. The sample was left for 15 min before being loaded into a capillary and imaged under the microscope. The dsRED channel (excitation FF01-554/23, emission FF01-609/54), and GFP channel (excitation FF01-474/27, emission FF01-525/45) were used for visualisation of the PI and Syto9 dyes, respectively.

Melaugh G., Martinez V.A., Baker P., Hill P.J., Howell P.L., Wozniak D.J, & Allen R.J. (2023). Distinct types of multicellular aggregates in Pseudomonas aeruginosa liquid cultures. NPJ Biofilms and Microbiomes, 9, 52.

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Microscopic Visualization of Bacterial Viability

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Microscopic images were obtained using a Zeiss LSM 780 NLO microscope. To visualize the bacterial strains, dyes were used. Prior to treatment with either violacein or B. bacteriovorus HD100, each of the bacterial cultures were mixed with 6 µM Syto-9 (Invitrogen, USA). This is a live stain and all viable bacterial cells were fluorescently green afterwards. After washing the cells with HEPES to remove any extra dye, they were exposed to either the predatory cells (PPR of 0.1) or 20 mg/ml of violacein in HEPES. After one hour, propidium iodide (Invitrogen, USA) was added to the bacterial cultures to a final concentration of 30 µM. This dye is a dead stain and labels any non-viable bacterial cells red. After 30 minutes at room temperature, the cells were pelleted (16,000 × g, 5 min), washed and resuspended in HEPES before being imaged.

Im H., Choi S.Y., Son S, & Mitchell R.J. (2017). Combined Application of Bacterial Predation and Violacein to Kill Polymicrobial Pathogenic Communities. Scientific Reports, 7, 14415.

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Multicolor Visualization of Biofilm Composition

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After biofilm cultivation in μ-dishes (see above), the supernatant was removed carefully and 1 ml of Brock medium (pH = 7.0) was used to wash the submersed biofilm. For staining, 2 ml of a fluorescent staining solution containing 250 μM of the DNA-binding dye SYTO9 (excitation: 483 nm, emission: 503 nm; Invitrogen, Thermo Fisher Scientific), 7.5 μg/ml of the fluorophore-labeled lectin concanavalin A (ConA)-Alexa 633 (excitation: 632 nm, emission: 647 nm, α-mannopyranosyl and α-glucopyranosyl residues; Invitrogen), and 15 μg/ml of GS-IB4-Alexa 568 (isolectin from Griffonia simplicifolia, excitation: 578 nm, emission: 603 nm, α-d-galactosyl and N-acetyl-d-galactosamine residues; Invitrogen) in Brock medium (pH = 7.0) were used. Samples were incubated for 30 min in the dark at room temperature. After staining, the supernatant was removed, the biofilm was washed twice with 1 ml of Brock medium (pH = 7.0) and finally 2 ml of Brock medium was added. Visualization was performed using a Zeiss LSM 510 laser scanning microscope with a 100× oil objective. Data processing was performed using the software Imaris 8.1.2 (Bitplane, Zürich, Switzerland).

Benninghoff J.C., Kuschmierz L., Zhou X., Albersmeier A., Pham T.K., Busche T., Wright P.C., Kalinowski J., Makarova K.S., Bräsen C., Flemming H.C., Wingender J, & Siebers B. (2021). Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress. Applied and Environmental Microbiology, 87(11), e02988-20.

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