Syto 63
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYTO 63 is a nucleic acid stain that can be used to fluorescently label DNA and RNA in various samples. It has an excitation maximum at 631 nm and an emission maximum at 652 nm, allowing it to be detected using the red fluorescence channel. SYTO 63 is membrane-permeant and can stain both live and dead cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Fluorescein isothiocyanate (fitc), by Yuanye Bio-Technology (1 mentions)
Fv1200, by Olympus (1 mentions)
Tcs sp5, by Leica (1 mentions)
Ab0576, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Brain heart infusion, by Merck Group (1 mentions)
Porcine gastric mucin type 2, by Merck Group (1 mentions)
Phosphate buffered saline solution, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Olympus (1 mentions)
7 protocols using syto 63
1
Multimodal Imaging of Sludge Microstructure
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Confocal laser scanning microscopy (CLSM) . CLSM (FV1200, Olympus, Japan) was employed to capture the images of the sludge samples before and after the pretreatment. SYTO 63 (Invitrogen, Carlsbad, USA), fluorescein isothiocyanate (FITC, Shanghai Yuanye Bio-Technology, China), concanavalin A (Con A, Xi'an Baiying Bio-Technology, China), and Calcofluor white (CW, Kulaibo Bio-Technology, China) were used to stain cells, proteins, α-d -glucopyranose polysaccharides, and β-D glucopyranose polysaccharides, respectively. Detailed operational procedures can be found in Text S1.
Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM-EDS) . To observe the sludge morphology before and after the pretreatment, the samples were fixed with 2.50% glutaraldehyde solution for 2 h. Then, they were dehydrated with ethanol and tert-butyl alcohol. The samples were freeze-dried and then imaged with a scanning electron microscope (SEM, SEISS Gemini 300). The EDS spectrometer (OXFORD Xplore) was used for the energy spectrum test.
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2
Biofilm Penetration Analysis by Microscopy
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The penetration of beads into the biofilm was quantified by confocal microscopy and image analysis. After exposure, the biofilms were fixed with 4% Paraformaldehyde (PFA) solution to preserve structure (von Ohle et al. 2010) . The fixed biofilms were stained with the nucleic acid stain Syto 63 (Invitrogen) to visualize the total biomass and subsequently imaged under Confocal Laser Scanner Microscope (CLSM, Leica TCS SP5). A non-treated biofilm was used as control. Confocal 3D stacks were collected f or each of three independent replicates for the non-treated, static, shaking and microspray experiments. For the biofilms exposed to the microsprays we took confocal images from three locations 1 to 2 mm outside of the clearance zone caused by the microspray (Supplemental material 1, Figure 1A, Appendix Figure 1A-B). For the 30 o shot we distinguished between the proximal area and distal areas with respect to distance from the device nozzle. For the non-treated, static and shaking exposures we collected data at 3 random positions on the slide (Supplemental material 1, Appendix Figure 1C). To quantify the penetration of the beads into the biofilm we used a relative depth ratio (RD BEADS ) to account for differences in biofilm thickness at each XY pixel location on the substratum, as explained previously (Miller et al. 2013 ) (Supplemental material 2).
3
Visualization of Biofilm Cellular Structure
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Following the growth of mat like biofilms on agar plates, 500 μL of 10 μM Syto 63 (S11345 from Thermo-fisher scientific) solution in Tris buffer was pipetted on the agar plate. The dye was allowed to stain the biofilms for 30 min. The biofilms were then scraped on the gene-frames (AB0576 from Thermo-fisher scientific) attached to a glass slide and sealed with a coverslip on the top. Images of the cellular structure were acquired with a Leica SP8 confocal microscope at a distance of 10–15 μm from the bottom glass coverslip using a ×100, 1.4 numerical aperture objective lens.
4
S. mutans Biofilm Growth and Characterization
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S. mutans UA159 (ATCC 700610) biofilms were grown for 72 h on glass microscope slides at 37°C and 5% CO2 in brain heart infusion (Sigma-Aldrich, St. Louis, MO) supplemented with 2% (wt/vol) sucrose (Sigma-Aldrich) and 1% (wt/vol) porcine gastric mucin (type II; Sigma-Aldrich). After the growth period, the biofilm-covered slides were gently rinsed in 1% (wt/vol) phosphate-buffered saline solution (Sigma-Aldrich) and placed in petri dishes (49 ). Biofilm thickness was determined by fixing untreated samples with 4% (wt/vol) paraformaldehyde and staining with Syto 63 (Thermo Fisher Scientific, UK). Subsequently, three random confocal images were taken on three independent replicate biofilm slides, with a thickness of 51.8 ± 4.9 μm measured using COMSTAT software; thus, we used a biofilm thickness (Lb) of 55 μm in the simulations.
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5
Biofilm Development Protein and Bacterial Staining
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Protein and bacterial staining was conducted according to previous study [30 (link)]. We monitored the changes in protein at a series of time points (2 h, 4 h, 8 h, 16 h, and 24 h) during the biofilm development in 24-well plate. 20 μM SYTO 63 (Molecular Probes, Eugene, OR, USA) was added to specimen for 30 min to stain bacteria (excitation/emission channel are 657 nm/673 nm); subsequently, the specimen was incubated with 100 μg/mL FITC (excitation/emission channel are 495 nm/525 nm) for 1 h to stain proteins. The bacteria were stained red while proteins were stained green. The biofilms formed on glass slides were examined by CLSM (Nikon A1, Nikon Corporation, Japan) with a 60x objective lens.
6
Confocal Microscopy of Biofilms with Kaempferol
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The confocal microscopy experiment was performed using confocal laser-scanning microscopy (Olympus, Shanghai, China). Syto 63 and fluorescein isothiocyanate (FITC) were purchased from Invitrogen Molecular Probes (Oregon). The biofilms were cultured according to the above method on glass cover slides with or without kaempferol (64 μg/ml). The biofilms produced by each group were washed with PBS after 12 h. Syto 63 (100 μM) was added to each well, and the plate was incubated with shaking for 5 min. FITC (0.001%) was then added, and the plate was incubated for another 30 min without shaking. The cells were washed with PBS to remove excess stain. Confocal microscopy images were obtained from NIS-Elements C version 3.2 (Nikon Eclipse).
7
Visualizing Microbial Cells on Carbon Felt
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At the end of the experiment, the carbon felt substratum was removed from each reactor and fixed onto a glass slide. To visualize total cells, the sample was mixed with 100 μL 20 μmol/L Syto 63 solution (Molecular Probes, Carlsbad, CA, USA) and incubated for 30 min [44] (link). The stained sample was then washed twice with phosphate-buffered saline (pH 7.2) to remove excess dye and imaged using a confocal laser scanning microscope (Carl Zeiss LSM710, Germany) equipped with a 100 × oil objective. Image processing and analysis were made using ZEN blue software (version 2012, Carl Zeiss, Germany).
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