Nuclear fragmentation assay was performed as described earlier [32 (link)]. Briefly, cells were seeded in 96-well plate at a density of (0.01 × 106 cell/200 μl/well) and kept at 37°C in CO2 incubator. Next day treatment was given (as described above) and after 24 h cells were terminated by centrifugation (100×g ; 10 min; 37°C). Cells were fixed in 96-well plate with 4% paraformaldehyde for 10 min followed by washing with PBS and permeabilization with 0.1% Triton X-100 (10 min). Further cells were washed with PBS and stained with DAPI (4,6-di-amidino-2-phenylindole) for 5 min. Images were captured using fluorescence microscope (Olympus IX 51 fluorescence microscope, Japan). 53 P-1 foci formation was performed in HEK transfected cells. The 53 P-1-GFP plasmid was a kind gift from Dr. Deepak Saini’s Laboratory, Indian Institute of Science (IISc), Bangalore, India. A permanently transfected cell line was established by transfecting this plasmid. For imaging, cells were seeded in 35-mm dishes containing coverslips at a density of (0.075 × 106 cells/2 ml/PD). Next day, treatment was given and cells were terminated at 4 and 24 h post treatment and images (GFP fluorescence) were captured to observe the foci formation using fluorescence microscope (Olympus IX 51 fluorescence microscope, Japan) under 10× (objective) × 10× (eyepiece) magnification.
Ix51 fluorescence microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany, Australia
The IX51 fluorescence microscope is a versatile instrument designed for optical microscopy. It features a high-performance illumination system and a robust mechanical structure, enabling consistent and reliable performance for a wide range of applications. The IX51 microscope is capable of fluorescence imaging, allowing users to visualize and analyze fluorescently labeled samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Xm10 camera, by Olympus (3 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Spark 10m, by Tecan (2 mentions)
Sybr green, by Bio-Techne (2 mentions)
Phosphate buffered saline (pbs), by Welgene (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Xm10 digital camera, by Olympus (2 mentions)
Hpa053669, by Merck Group (2 mentions)
Elastin, by Merck Group (1 mentions)
Elastic stain kit, by ScyTek Laboratories (1 mentions)
4 6 diamidino 2 phenyindole dapi, by Vector Laboratories (1 mentions)
Edu proliferation assay kit, by RiboBio (1 mentions)
Cck 8, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Solarbio (1 mentions)
121 protocols using ix51 fluorescence microscope
1
Quantifying Nuclear Fragmentation and 53BP1 Foci
2
Immunofluorescence Staining of Cultured Cells
3
Immunofluorescence Staining of Cultured Cells
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Cultured cells were fixed with 4% PFA for 20 min and made permeable using 0.4% Triton X-100 in PBS for 20 min. Cells were treated with 3% BSA in PBS for 1 h at room temperature followed by overnight incubation with the primary antibody at 4 °C. Cells were rinsed with PBS 3 X 5 min and incubated with secondary antibody for 1 h at room temperature. Control and experimental wells were processed identically.
Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Images were taken using IX51 Olympus fluorescence microscope and merged and quantified using Image J software. Antibodies used are listed inTable 3 .
Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Images were taken using IX51 Olympus fluorescence microscope and merged and quantified using Image J software. Antibodies used are listed in
4
Quantifying Virus Titers in BSR Cells
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A direct fluorescent antibody test was performed to determine virus titers in BSR cells. BSR cells were incubated with a ten-fold serially diluted rRABVs in 96-well plates at 37 °C for 48 h. Subsequently, the incubated cells were washed with 300 μL phosphate-buffered saline (PBS) three times, then fixed with 80% ice-cold acetone and stained with FITC-conjugated RABV N protein-specific antibodies for 1 h. Antigen-positive fluorescent spots were counted as focus-forming units per milliliter (FFU/mL) under Olympus IX51 fluorescence microscope (Olympus, Tokyo, Japan). Virus dilution at each concentration was added into four wells.
5
Elastic Matrix Characterization of Tissue Constructs
6
Pleuromutilin Inhibits A2780 and Caov-3 Cell Proliferation
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A2780 and Caov-3 cell proliferation after 48 of treatment with pleuromutilin was determined using Edu proliferation assay in accordance with the manufacturer’s instructions. The cells were distributed at 3×104 cells/well density in 96-well plates and cultured for 24 h. The medium was changed by fresh medium mixed with 10, 20, 40, 80, 160, and 200 μM of pleuromutilin, and incubation was performed for 48 h. Cell proliferation was determined using an Edu proliferation assay kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China). An Olympus IX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) was used to observe the stained cells.
7
Assessing HUVEC Cell Proliferation
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CCK-8 (Sigma-Aldrich, USA) assay was carried out to detect the proliferation of HUVEC cells. Ox-LDL-treated HUVEC cells (1 × 103 cells per well) were seeded in the 96-well plates. Then 10 μL CCK-8 reagent was added and incubated for 45 min, and the absorbance was detected at 450 nm.
For the EdU assay, transfected ox-LDL-treated HUVEC cells were incubated with 10 μM EdU reagents for 2 h in the dark. Cells were then fixed in phosphate buffer saline (PBS, containing 4 % paraformaldehyde (PFA; Sigma-Aldrich), Thermo Fisher Scientific) for 15 min. Subsequently, 2 mg/mL glycine (Solarbio, Beijing, China) and 0.5 % Triton X-100 (Solarbio) were added to each well. After that, DAPI (Vector Laboratories) was added to each well. Then, cells were incubated in the dark for 30 min. Images were observed using a fluorescence microscope (Olympus IX 51 fluorescence microscope, Japan) [26 (link)].
For the EdU assay, transfected ox-LDL-treated HUVEC cells were incubated with 10 μM EdU reagents for 2 h in the dark. Cells were then fixed in phosphate buffer saline (PBS, containing 4 % paraformaldehyde (PFA; Sigma-Aldrich), Thermo Fisher Scientific) for 15 min. Subsequently, 2 mg/mL glycine (Solarbio, Beijing, China) and 0.5 % Triton X-100 (Solarbio) were added to each well. After that, DAPI (Vector Laboratories) was added to each well. Then, cells were incubated in the dark for 30 min. Images were observed using a fluorescence microscope (Olympus IX 51 fluorescence microscope, Japan) [26 (link)].
8
Cytotoxicity Assessment of PEG-PLGA Nanoparticles
9
Fluorescent Antibody Virus Neutralization Assay
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Blood samples of the mice were collected at different time points post immunization, and the level of VNA was determined by fluorescent antibody virus neutralization (FAVN) tests. The prepared 50 μL three-fold diluted experiment samples and standard serum samples (National Institute for Biological Standards and Control, Herts, UK) were added into 96-well plates. Each well was added with 50 μL CVS-11 suspension containing 100 FFU, and the samples were incubated at 37 °C, 5% CO2 for 1 h, added with 50 μL BSR cells (5 × 105 cells/mL) and incubated at 34 °C, 5% CO2 for three days. Then each well was added with 100 μL ice cold acetone (pre-cooled at −20 °C) and incubated at room temperature for about 30 min and air-dried. Afterwards, the samples were stained with FITC-conjugated anti-RABV N antibodies for 45 min at 37 °C, and then washed three times with PBS. The resultant samples were observed under an Olympus IX51 fluorescence microscope (Olympus, Tokyo, Japan). We compared the fluorescence values of our prepared serum samples with those of standard samples to obtain VAN titers, which were expressed as international units per milliliter (IU/mL).
10
Cytotoxicity of TGF-β1 and DOX Loaded PLGA NPs
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