Following the exposure to the above solutions, all specimens were rinsed gently in 0.85% physiological saline and then stained with a 1:1 mixture of SYTO 9 and propidium iodide (BacLight LIVE/DEAD Bacterial Viability kit, Molecular Probes, Eugene, OR) following the manufacturer's instructions. Images of the stained samples were taken by a CSLM (FV10i-LIV, Olympus, Canada) at 480/500 nm for SYTO 9 and 490/635 nm for propidium iodide, respectively. Five random areas were chosen from each specimen and scanned at a resolution of 512 × 512 pixels with 2 μm step size from the top to the bottom of the biofilm for each chosen area. Three samples were observed for each group and five randomly selected areas of each sample were scanned. Three-dimensional volume stacks were reconstructed with Imaris 7.2 software (Bitplane Inc., St Paul, MN, USA), and the total volume (red and green fluorescence) was measured. The proportion of dead bacteria was indicated by the proportion of red fluorescence of the total of green and red fluorescence.
Fv10i liv
Manufactured by Olympus
Sourced in Japan, Canada
The FV10i-LIV is a laser scanning confocal microscope system designed for live-cell imaging. It provides high-resolution, real-time imaging capabilities to support a variety of biological research applications.
Automatically generated - may contain errors
Lab products found in correlation
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (2 mentions)
Uplansapo 60x na 1, by Olympus (1 mentions)
Blocking solution, by Nacalai Tesque (1 mentions)
Nucblue fixed cell stain readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal anti ha antibody, by Merck Group (1 mentions)
Mouse monoclonal anti v5 antibody, by Merck Group (1 mentions)
Alexa fluor 594 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Biotium (1 mentions)
Softmax pro 5, by Molecular Devices (1 mentions)
35 mm glass bottom dishes, by AGC Techno Glass (1 mentions)
11 protocols using fv10i liv
1
Quantitative Live-Dead Biofilm Analysis
2
Fluorescent Lipid Delivery to Mitochondria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Empty MITO-Porters containing 0.5 mol% NBD-DOPE (green fluorescent lipid) were prepared by the lipid hydration method, as previously reported.10 (link),29 (link) Cells were seeded in 35 mm glass-bottom dishes (AGC Techno Glass [IWAKI], Shizuoka, Japan), 24 h prior to the experiment (2 mL, 8 × 104 cells/mL, incubation at 37°C, 5% CO2). After washing the cells twice with 1 mL DMEM (FBS−), they were incubated with DMEM that did not contain FBS and in the presence of the MITO-Porter (10 nM lipid) for 1 h. The medium was removed, and 1 mL of DMEM (FBS+) was added. After incubation for a further 1 h and 40 min, the cells were stained with MitoTracker Deep Red (1 mL, final concentration 100 nM), 20 min prior to observation. Cells were washed with DMEM (phenol red−), and 1 mL of fresh DMEM (phenol red−) was added before CLSM images were obtained using a FV10i-LIV (Olympus, Tokyo, Japan). The cells were excited with a 473-nm light and a 635-nm light from an laser Diode (LD) laser. Images were obtained using an FV10i-LIV equipped with a water-immersion objective lens (UPlanSApo 60×/numerical aperture [NA] 1.2) and a dichroic mirror (DM405/473/559/635). The two fluorescence detection channels (Ch) were set to the following filters: Ch1: 490/50 (green) for NBD-DOPE; Ch2: 660/50 (red) for MitoTracker Deep Red.
3
Intratumoral rTPA-MITO-Porter Light Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mice bearing SAS cells were intratumorally injected with the rTPA–MITO-Porter (rTPA dose = 8.2 μg per mouse) or an HBG solution (as negative control), followed by irradiation with 700 nm light for 20 min, at 12 h after drug administration. The mice were sacrificed 3 h after the light irradiation process, and tumor tissues were collected. The tumor tissues were incubated with 0.5 μM MitoProbe™ TMRM solution in PBS (−) for 30 min, followed by observation using a CLSM. The Olympus FV10i-LIV is equipped with a water-immersion objective lens (UPlanSApo 60X/NA. 1.2), and a dichroic mirror (DM405/473/559/635) was used to detect the TMRM signal. The tissues were illuminated with 559 nm light to excite the TMRM, and the fluorescence detection channel was set using a filter at a bandpass 570–670 nm to detect the TMRM fluorescence signal. The quantification of the mean fluorescence intensity of the TMRM was then performed from randomly selected CLSM images using the ImageJ software.32 (link)
4
Dynamic Microscopic Imaging of Cell-Bead Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal imaging was conducted at 48 h after the seeding of the cells. We used FV10i-LIV (Olympus) to simultaneously acquire phase contrast images of the cells and fluorescent images of the beads. The trial period lasted for 6-10 h and the sampling rate was one frame per 5 min. After each trial, we removed the cells by the trypsinization and imaged the strain-free pattern of the fluorescent beads.
5
Localization of Overexpressed Proteins in SH-SY5Y Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the location of APP770-V5 and FNDC5-HA, we examined immunofluorescence staining of SH-SY5Y cells transiently expressing these molecules. We washed prepared cells by PBS and fixed them by 4% paraformaldehyde for 15 min at room temperature. Then, these cells were permeabilized by 0.1% Triron-X and blocked by using Blocking solution (Nacalai Tesque, Japan). We used the mouse monoclonal anti-V5 antibody (1:1000; Sigma) and the rabbit polyclonal anti-HA antibody (1:1000; Sigma) for the primary antibodies to detect APP and FNDC5, and then labeled them by Alexa Fluor 594-conjugated goat anti-mouse (1:2000; Life Technologies, MA, USA) and Alexa Fluor 488-conjugated mouse anti-rabbit (1:2000; Life Technologies), respectively. As the mounting agent, we used NucBlue Fixed Cell Stain ReadyProbes reagent from Life Technologies. These cells were observed using a laser confocal scanning microscope (FV10i-LIV, Olympus, Japan).
6
Quantifying Live and Dead Bacteria on Titanium Disks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After incubation for one or two 48-h periods, titanium disks coated with biofilms were gently washed with PBS and stained with a fresh mixture of SYTO-9 and propidium iodide (BacLight live/dead bacterial viability kit, Molecular Probes, Eugene, United States) based on the manufacturer’s instruction. And observed by confocal laser scanning microscopy (CLSM, FV10i-LIV, Olympus, Canada) (Yu et al., 2021 (link); Liu et al., 2022 (link)). The wavelength of excitation/emission light detected for SYTO-9 and propidium iodide was set 480/500 nm and 490/635 nm, respectively. For each group, three disks were selected, and five areas for each disk were scanned from the bottom to the top of the biofilm with a 2 μm step. An Imaris 7.2 software (Bitplane, Switzerland) was employed to convert the two-dimension images into three-dimensional volume stacks. The obtained green and red fluorescence intensity was analyzed as the total volume of live and dead bacteria, and the dead bacteria proportion was calculated using red fluorescence in relation to total fluorescence (green + red) (Guo et al., 2021 (link)).
7
JC-1 Assay for Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with samples in a 96-well white plate for 24 h before adding 25 μM of 3-NP. MMP of PC12 cells was measured by using a JC-1 mitochondrial membrane potential assay kit (Biotium, Hayward, CA, USA) according to the manufacturer’s instructions. The plates were then incubated at 37°C for 20 min after the addition of 100 μl of 1 × JC-1 reagent into the wells. Red fluorescence (excitation, 550 nm; and emission, 600 nm) and green fluorescence (excitation, 485 nm and emission, 535 nm) were determined using a Softmax Pro 5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). The ratio of red to green fluorescence of dead cells and of cells undergoing apoptosis is lower than that of the healthy cells. For confocal microscope analysis, 1× JC-1 was added to treated cells and cells were incubated for 15 min at 37°C. Cells were imaged using a confocal microscope (FV10i-LIV, Olympus, Tokyo, Japan). In live non-apoptotic cells, mitochondria appeared red following aggregation of the JC-1 reagent. The excitation of the red aggregates occurred at 559 nm and emission was at 570–620 nm. In both the apoptotic cells and dead cells, the dye remained in its monomeric form and appeared green with excitation at 473 nm and emission at 490–540 nm.
8
Immunofluorescence Staining of Gangliosides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen sections were blocked with 1% BSA in PBS containing 0.075% Tween‐20 for 1 hour. After washing with PBS, sections were incubated with primary Abs diluted in 1% BSA in PBS containing 0.01% Tween‐20 for 1 hour. Then sections were washed with PBS and incubated with secondary Abs diluted in 1% BSA containing 0.01% Tween‐20 for 1 hour. After washing with PBS, counterstaining with DAPI was carried out, and sections were mounted in 90% glycerol. Expression patterns of gangliosides were observed under confocal microscopy (FV10i‐LIV; Olympus Life Science).
9
Imaging of Mitochondrial Delivery Vehicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The empty-MITO-Porter and mRNA-MITO-Porter, labelled with 0.1 mol% DiI, were prepared by the ethanol dilution method. At 24 hrs before being observed, the cells (5 × 104 cells) were seeded in 35 mm glass-bottom dishes (AGC TECHNO GLASS CO., LTD. (IWAKI), Shizuoka, Japan) at 37 °C under 5% CO2. After washing the cells twice with 1 mL of DMEM (FBS-), they were incubated with DMEM (FBS-) that contained the MITO-Porters (300 ng RNA). After a 1 hr period of incubation, the medium was removed and 1 mL of DMEM (FBS+) was added. After the cells were incubated for 1 hr and 40 min, DMEM was then removed, and the cells were stained with MTDR in DMEM (1 mL, final concentration of 100 nM) for a further 20 min. After washing the cells with 1 mL DMEM (phenol red -), 2 mL of DMEM (phenol red -) was added. Cells were analyzed by CLSM using FV10i-LIV (Olympus Corporation, Tokyo, Japan), based on the experimental section of the paper we previously reported47 (link). The cells were excited with a 559 nm light from an LD laser. Images were obtained using an FV10i-LIV equipped with a water-immersion objective lens (UPlanSApo 60×/NA. 1.2) and a dichroic mirror (DM405/473/559/635). The two fluorescence detection channels (Ch) were set to the following filters: Ch1: 570/50 (red) for DiI, Ch2: 660/50 (red) for MTDR.
10
Visualizing Mitochondria and Plasmid Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
(1 × 10 5 cells/dish) were seeded on a 3.5 cm glass-base dish (Iwaki; ASAHI GLASS Company, Ltd., Tokyo, Japan) at one day before transfection and incubated in DMEM, which contained 10% FBS, under an atmosphere of 5% CO2/air at 37 °C. The cells were incubated with carriers containing 1-μg Cy-5 labeled pCMV-mtLuc (CGG) suspended in 1 mL of phenol red-free medium without FBS, followed by incubation under 5% CO2 at 37ºC. After 1-hr incubation, the medium was replaced with fresh phenol red-free medium containing FBS, and the cells were incubated for 2 hr. The medium was replaced with fresh medium containing MitoFluor Red 589 (final concentration, 100 nM) 20 min before the acquisition of the fluorescence images, and the cells were incubated in this solution. After this incubation, the cells were washed with the phenol redfree medium containing FBS, and then observed by CLSM (FV10i-LIV; Olympus Corporation, Tokyo, Japan). The cells were excited with a 559 nm light for detecting MitoFluor Red 589 and a 635 nm light for detecting Cy5 using a LD laser. Images were obtained using a FV10i-LIV equipped with a water immersion objective lens (UPlanSApo 60x/NA = 1.2) and a diachronic mirror (DM 405/473/559/635). The two fluorescence detections Chs were set to the following filters: Ch1: BP 570-620 (red color) for MitoFluor Red 589 and Ch2: BP 660-710 (green pseudo color) for Cy5.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!