The number of LC3 puncta and colocalization of LC3 with acidified lysosomes was determined by confocal microscopy as previously described61 (link). Briefly, BMM cultured on cover slips were infected with IOE at MOI of 5 or left uninfected. Cells were then washed 3X with PBS, fixed with 2% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After blocking with 5% BSA (Sigma-Aldrich, A2153) for 60 min, the primary antibodies; anti-LC3 (Sigma, 50 ug/mL) was added for 1 h at room temperature. Cells were washed and then incubated with fluorescent labeled anti-rabbit secondary antibody DyLight (VectaFluor, 1:500) for 1 h. Nuclei were stained with DAPI and cells were analyzed by confocal microscopy (Olympus Flouview 1000). Analysis of acidified lysosome was performed using LysoTracker Red (cat. L-12492, Thermofisher) at 37 °C for 1 h and assessed with a confocal microscope (Olympus Flouview 1000).
Flouview 1000
Manufactured by Olympus
Sourced in United States
The FlouView 1000 is a fluorescence imaging system designed for biological and materials science research. It provides high-resolution, confocal imaging capabilities for a wide range of applications.
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6 protocols using flouview 1000
1
Quantifying LC3 Puncta and Lysosomal Colocalization
2
Automated Fluorescence Microscopy Imaging
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An Olympus FlouView 1000 confocal laser scanning microscope fitted with 3 lasers was used. GFP was excited using the 488 nm laser line, the 543 nm line was used for EBr, and the 633 nm line for excitation of Alexa Fluor 633. Microscopy images were taken using the 60x oil immersion objective (NA = 1.35) analysed using Fiji ImageJ.
Automated microscopy was done using an iCys Research Imaging Cytometer (CompuCyte Corporation, Westwood, MA). The instrument is based on an Olympus IX-71 inverted microscope equipped with four lasers, photodiodes (detecting light loss and scatter) and four photomultiplier tubes (PMTs). A 405 nm solid state laser was used for the excitation of DAPI and Hoechst, the 488 nm Argon laser line was used to excite GFP and EBr, and a 633 nm HeNe laser for Alexa 633. For each salt treatment, 1500–2000 events were measured. Data analysis was performed using the iCys 7.0 software, and graphs were prepared using SigmaPlot 14.0.
3
Boeravinone B Potentiates Biofilm Disruption
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The potentiating effect of boeravinone B was further confirmed by staining the biofilms with FITC and DAPI which stains protein (amine reactive) and nucleic acid (DNA), respectively, by modified method as described by Yang (using DAPI in place of Hoechst) followed by visualization by confocal laser scanning microscopy (Yang et al., 2015 (link)). The biofilm of S. aureus ATCC 29213 was grown in six well poly styrene plate (Nunc) containing sterile 18 mm glass cover slips. Ciprofloxacin was used at 4 μg/ml which is MBIC and at 1 μg/ml which is subMBIC (0.25× MBIC) as determined above. Boeravinone B at 12.5 μM (MBIC) was added to sub-inhibitory concentration of ciprofloxacin (0.25× MBIC). The plates were incubated for 24 h at 37°C. PBS washes were given to eliminate planktonic cells followed by fixation of biofilm with 5% para-formaldehyde for 1 h at 50°C. Fixed biofilm was flooded with 0.001% (w/v) FITC and 1 μg/ml DAPI at kept at room temperature for 1 h. Images were seen and captured on 40× oil immersion lens using CLSM (Olympus flou View 1000).
4
Intravital 2-P Microscopy Imaging Protocol
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Intravital 2-P microscopy was performed as previously reported.18 (link) Initial studies were conducted on our custom-built Olympus Flouview-1000 using a ×60 water immersion lens (numerical aperture 1.2). Later studies were conducted using a Leica Dive Multiphoton system, using a ×40 water immersion objective (numerical aperture 1.1).16 (link) During image acquisition, changes in laser power transmissivity were used to assure minimal saturation of endocytic accumulation to allow correct quantitation of accumulation,25 (link) with the exception of the TR-RAP/CBdex study, where the 40-mg/kg dose required for pharmacological efficacy caused signal saturation. For this reason, no quantitative analysis was conducted with these data. A laser transmissivity compensation curve was generated to normalize the 12-bit intensity readings for the most accurate analysis of accumulation; this has been characterized on both microscopy systems. Rats were anesthetized using isoflurane (5% induction, 1.5%–2.0% maintenance at 0.5 L/min of O2) and monitored as previously described.26 (link)
5
Monitoring Bacterial Attachment and Biofilm
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To monitor initial surface interaction and attachment, E. amylovora strains expressing pMP2444::gfp [13 (link)] were grown for 18 h at 28°C and normalized to an OD600 of 0.5. A total of 1 ml of inoculum for each strain was introduced into a flow cell chamber in a μ-Slide VI 0.5 glass bottom slide (Ibidi, Martinsried, Germany). Immediately, the base of the flow chamber was repeatedly imaged using a Olympus FlouView 1000 confocal laser scanning microscope (Olympus, MA, USA). Images were acquired for up to 1 h or until the frame was saturated with fluorescent cell signals. Following this, the flow cell chamber was flushed with 5 ml of 0.5X phosphate buffered saline (PBS). To evaluate biofilm formation, following the initial attachment incubation, the flow chamber was subjected to flow (0.5X LB) using a peristaltic pump (Ismatec REGLO Digital 4-CH pump) (Cole-Parmer IL, USA) for 5 h. Fluorescent Z-stacked images were acquired to measure overall attachment and biofilm levels in the flow cell chambers [13 (link)]. ImageJ software was used to invert the color on the images, and the RBG plugin was used to process these images and to graph the GFP signal intensity profile for the Z-stacked images [62 (link)].
6
Monitoring Bacterial Surface Attachment
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To monitor initial surface interaction and attachment, E. amylovora strains expressing pMP2444::gfp (16) were grown for 18 h at 28°C and normalized to an OD600 of 0.5. A total of 1 ml of inoculum for each strain was introduced into a flow cell chamber in a µ-Slide VI 0.5 glass bottom slide (Ibidi, Martinsried, Germany). Immediately, the base of the flow chamber was repeatedly imaged using a Olympus FlouView 1000 confocal laser scanning microscope (Olympus, MA, USA). Images were acquired for up to 1 h or until the frame was saturated with fluorescent cell signals. Following this, the flow cell chamber was flushed with 5 ml of 0.5X phosphate buffered saline (PBS). To evaluate biofilm formation, following the initial attachment incubation, the flow chamber was subjected to flow (0.5X LB) using a peristaltic pump (Ismatec REGLO Digital 4-CH pump) (Cole-parmer IL, USA) for 5 h. Fluorescent Z-stacked images were acquired to measure overall attachment and biofilm levels in the flow cell chambers (16) . ImageJ software was used to process these images and to graph the GFP signal intensity profile for the Z-stacked images (39) .
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