Cell morphology and proliferation were assessed at days 1, 3, and 5 after seeding. For cell morphology assessment, live cells were stained with Calcein AM (2 μM) and cell nuclei were stained with Hoechst 33342 (1:1000). The stained cells were observed using Olympus FluoView FV1000 confocal microscope (Carl Zeiss, Oberkochen, Germany). Cell proliferation was measured using Quant-iT PicoGreen dsDNA assay kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s recommendations. First, the total DNA content was extracted and purified from the samples using QIAamp DNA mini kit (Qiagen, Valencia, CA, USA). The DNA content solution was then stained with PicoGreen fluorescent nucleic acid dye. The relative fluorescence intensity was quantitatively measured at 480 nm excitation and 520 nm emission using a Synergy H1 hybrid microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Equal number of cells cultured under same conditions were used as controls. All experiments were performed in triplicate.
Fluoview fv1000 confocal microscope
Manufactured by Zeiss
Sourced in Germany
The FluoView FV1000 is a confocal microscope designed for high-resolution imaging. It features laser-based illumination, multiple detection channels, and advanced software for image acquisition and analysis. The core function of the FluoView FV1000 is to provide researchers with a tool for detailed, high-quality imaging of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (8 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Fluorescent secondary antibody, by Cell Signaling Technology (2 mentions)
Anti ubiquitin, by Abcam (2 mentions)
Anti cox 4, by Abcam (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti cox 4, by Cell Signaling Technology (2 mentions)
Mitosox red mitochondrial superoxide indicator, by InvivoGen (2 mentions)
Synergy h1 hybrid microplate reader, by Agilent Technologies (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Voltalef 10s oil, by Avantor (1 mentions)
Deltavision microscope, by Cytiva (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Metamorph microscopy automation and image analysis software, by Molecular Devices (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Kma 0100 00a, by CellPath (1 mentions)
10 protocols using fluoview fv1000 confocal microscope
1
Cell Morphology and Proliferation Assessment
2
FRET Imaging of Virus-Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were excited by a 405-nm laser diode, and CFP and Venus were detected at 480–500 nm and 515–615 nm wavelength ranges, respectively. The images were obtained using an Fluoview
FV1000 confocal microscope with an oil-immersion UPLSAPO objective (NA=1.35) (Carl Zeiss, Jena, Germany). Using the FRET application, the fluorescence intensities from CFP and Venus channels
were obtained from individual fluorescence-expressing cells. All image analyses were performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).
The fluorescence intensities of cytoplasmic areas in cells were obtained manually by tracing the cytoplasm, using drawing tablet (Wacom) for BVDV, EMCV, and JEV-infected cells. The
corrected total cell fluorescence (CTCF) from Venus and CFP channel was then calculated using the formula: Integrated Density−(Area of selected cell) × (Mean fluorescence of background
readings). Each Venus/CFP emission ratio was calculated by dividing the CTCF of a Venus image with a CFP image.
FV1000 confocal microscope with an oil-immersion UPLSAPO objective (NA=1.35) (Carl Zeiss, Jena, Germany). Using the FRET application, the fluorescence intensities from CFP and Venus channels
were obtained from individual fluorescence-expressing cells. All image analyses were performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).
The fluorescence intensities of cytoplasmic areas in cells were obtained manually by tracing the cytoplasm, using drawing tablet (Wacom) for BVDV, EMCV, and JEV-infected cells. The
corrected total cell fluorescence (CTCF) from Venus and CFP channel was then calculated using the formula: Integrated Density−(Area of selected cell) × (Mean fluorescence of background
readings). Each Venus/CFP emission ratio was calculated by dividing the CTCF of a Venus image with a CFP image.
3
Visualizing Plant-Microbe Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the gnotobiotic system, the plants were taken out from the semisolid agar medium after inoculation for certain times. Plant root surfaces were rinsed with sterile water and then were directly observed under the laser confocal (Olympus FluoView™ FV1000 confocal microscope or Zeiss LSM 710 confocal microscope). Some tissues of roots, stems and leaves were sectioned in longitudinal and transverse directions to examine the colonization of GFP-labelled P. polymyxa WLY78. In the soil system, the plants were taken out from the soil, rinsed with sterile water and observed directly under the laser confocal. The resulting images were obtained using the FV10-ASW 4.0 Viewer software or Zen 2012 software.
4
Live and Fixed Ovary Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For live imaging, ovaries were dissected and imaged in Voltalef oil 10S (VWR International) on either a widefield DeltaVision microscope (Applied Precision, WA, USA) equipped with a Photometrics 512 EMCCD camera (Photometrics, AZ, USA) and a 2x magnification tube fitted between the unit and the camera, or an Olympus Fluoview FV1000 confocal microscope (Olympus, Japan) or on an Olympus IX81 inverted microscope with a Yokogawa CSU22 spinning disk confocal imaging system using 100 × 1.4 NA Oil UPlanSApo objective lens (Olympus, Japan). Fixed preparations were imaged using an Olympus Fluoview FV1000 confocal microscope (40 × 1.35 NA Oil UPlanSApo, 60 × 1.35 NA Oil UPlanSApo) or Zeiss LSM510 Meta laser scanning confocal system (Carl Zeiss Microimaging, Inc.) with Plan-Neofloar 40x (Oil) NA1.3 and Plan-Apochromat 63x (Oil) NA1.4 objectives. Images were collected with the softWorXs software (Applied Precision), Olympus Fluoview (Olympus, Japan), MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices, CA, USA, RRID:SCR_002368 ) or Zeiss LSM 510 AIM software (Carl Zeiss Microimaging, Inc.) and processed using Fiji (Fiji, RRID:SCR_002285 ) (Schindelin et al., 2012 (link)).
5
Quantification of Intestinal Organoid Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoid images were taken using a Keyence BZ9000 microscope. Intestinal tissue sections were imaged using either a Olympus Fluoview FV-1000 confocal microscope or a Zeiss LSM780 confocal microscope. Images were processed using Adobe Photoshop CS5. Antibody-positive cells were quantified as the total number of labeled cells/total number of DAPI+ nuclei present in a crypt section. Twenty crypts were sampled per animal for the quantification of Ki67, phosphorylated histone H3 (pHH3), phosphorylated c-JUN (pJUN), Caspase3, or FUCCI reporters, with 3–4 animals/organoid lines examined per condition. Paneth cells were quantified as the number of lysozyme-positive cells per crypt in 30 crypts sampled from 3 animals of each genotype. Goblet cells were quantified as the number of alcian blue–positive cells in 10 villi field of views (×20 objective) per animal from 3 animals of each genotype. Crypts were counted by sampling 10 regions of 1 mm from 6 animals of each genotype. Quantification of data shown in Figures 1 B, 2 A, 3 A, and 3 C was done at the time point ZT4.
6
Imaging Autophagy and Mitochondria in Yersinia pestis Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on a glass-bottomed cell culture dish (801002, NEST, Wuxi, Jiangsu, China) in the indicated medium and infected with Y. pestis as described above (MOI = 40 for 4 h) or treated with CCCP at the indicated time points. Cells were then washed with PBS and fixed for 15 min at room temperature with 4% paraformaldehyde in PBS, followed by three washes with PBS. After permeabilization with 0.2% Triton X-100 in PBS for 10 min and blocking with 5% BSA in PBS for 1 h, cells were incubated with primary antibodies, including anti-LC3B (1:200, 3868, Cell Signaling Technology, Danvers, MA), anti-COX IV (1:500, ab33985) and anti-Ubiquitin (1:500, ab223378) (Abcam, Boston, MA), and anti-COX IV (1:500, 4850, Cell Signaling Technology) overnight at 4°C. Cells were then washed three times with PBS and incubated for 1 h at room temperature with fluorescent secondary antibody (Cell Signaling Technology), followed by three washes with PBS, incubation with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and observation with the Olympus FluoView FV1000 confocal microscope or the Zeiss LSM 880 confocal microscope.
7
Mitochondrial Superoxide Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MitoSOX staining was performed following the manufacturer’s protocol (Invivogen). Cells were cultured on a glass-bottomed cell culture dish in the indicated medium and infected with Y. pestis as described above (MOI = 40 for 4 h) or treated with CCCP at the indicated times. Then, the cells were incubated with 5 μM MitoSOX Red mitochondrial superoxide indicator (M36008, Invivogen, San Diego, CA) in cell culture medium at 37°C for 10 min, protected from light. After washing with PBS, the cells were incubated with DAPI for 5 min and observed with the Olympus FluoView FV1000 confocal microscope or the Zeiss LSM 880 confocal microscope.
8
Mitochondrial Superoxide Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MitoSOX staining was performed following the manufacturer’s protocol (Invivogen). Cells were cultured on a glass-bottomed cell culture dish in the indicated medium and infected with Y. pestis as described above (MOI = 40 for 4 h) or treated with CCCP at the indicated times. Then, the cells were incubated with 5 μM MitoSOX Red mitochondrial superoxide indicator (M36008, Invivogen, San Diego, CA) in cell culture medium at 37°C for 10 min, protected from light. After washing with PBS, the cells were incubated with DAPI for 5 min and observed with the Olympus FluoView FV1000 confocal microscope or the Zeiss LSM 880 confocal microscope.
9
Imaging Autophagy and Mitochondria in Yersinia pestis Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on a glass-bottomed cell culture dish (801002, NEST, Wuxi, Jiangsu, China) in the indicated medium and infected with Y. pestis as described above (MOI = 40 for 4 h) or treated with CCCP at the indicated time points. Cells were then washed with PBS and fixed for 15 min at room temperature with 4% paraformaldehyde in PBS, followed by three washes with PBS. After permeabilization with 0.2% Triton X-100 in PBS for 10 min and blocking with 5% BSA in PBS for 1 h, cells were incubated with primary antibodies, including anti-LC3B (1:200, 3868, Cell Signaling Technology, Danvers, MA), anti-COX IV (1:500, ab33985) and anti-Ubiquitin (1:500, ab223378) (Abcam, Boston, MA), and anti-COX IV (1:500, 4850, Cell Signaling Technology) overnight at 4°C. Cells were then washed three times with PBS and incubated for 1 h at room temperature with fluorescent secondary antibody (Cell Signaling Technology), followed by three washes with PBS, incubation with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and observation with the Olympus FluoView FV1000 confocal microscope or the Zeiss LSM 880 confocal microscope.
10
Cryopreservation and Immunostaining of Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids were fixed in 4% PFA at 4 °C for 10 min, washed in PBS and transferred to 30% sucrose solution overnight at 4 °C. Samples were embedded in tissue-tek (CellPath, KMA0100-00A) snap-frozen on dry ice and stored at –80 °C. Twenty micrometersections were obtained with a cryostat.
Sections were blocked for 1 h in blocking solution (1% Triton-X, 3% BSA and 10% donkey or goat serum) at RT followed by incubation with primary antibodies in blocking solution overnight at 4 °C. Sections were then washed with PBS and incubated with secondary antibodies and Hoechst (ThermoFisher, H3569) at RT for 2 h. When Hoechst staining was not performed, samples were incubated with DAPI for 5 min before mounting. See Supplementary Table4 for antibodies used in this study. Samples were mounted on glass coverslips using Fluorosave (CalBioChem, 345789) and imaged (Olympus (Tokyo, Japan) Fluoview FV1000 confocal microscope or Zeiss Axio- Scope A1). In all, ≥ 3 organoids from three batches were sectioned and imaged.
Sections were blocked for 1 h in blocking solution (1% Triton-X, 3% BSA and 10% donkey or goat serum) at RT followed by incubation with primary antibodies in blocking solution overnight at 4 °C. Sections were then washed with PBS and incubated with secondary antibodies and Hoechst (ThermoFisher, H3569) at RT for 2 h. When Hoechst staining was not performed, samples were incubated with DAPI for 5 min before mounting. See Supplementary Table
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!