The day prior to infection, 50,000 VeroE6 cells per well were seeded in 96-well tissue culture plates using 10% FBS DMEM, and then incubated overnight at 37 °C in a humidified, 5% CO2 atmosphere-enriched chamber. Serial 2.5-fold dilutions (from 10−1 to 10−6.5) of the samples were prepared in DMEM and used to infect Vero E6 cells; each dilution was tested in four replicates. The plates were incubated for at least 96 h and observed to monitor the development of cytopathic effect using an EVOS Floid microscope (Invitrogen). Viral titres, expressed as median tissue culture infectious dose (TCID50/mL), were calculated according to both Reed and Muench and Karber methods based on three or four replicates for dilution [66 (link)].
Evos floid microscope
Manufactured by Thermo Fisher Scientific
The EVOS Floid microscope is a compact, all-in-one fluorescence and bright-field imaging system designed for routine cell culture observation and analysis. It features high-quality optics, a user-friendly interface, and a compact footprint, making it suitable for a variety of laboratory applications.
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Lab products found in correlation
5 protocols using evos floid microscope
1
SARS-CoV-2 Viral Titration in VeroE6 Cells
2
Viral Titer Quantification in VeroE6 Cells
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The day prior to infection, 50,000 VeroE6 cells per well were seeded in 96-well tissue culture plates using 10% FBS DMEM, and then incubated overnight at 37 ◦C in a humidified, 5% CO2 atmosphere-enriched chamber. On the day of infection, serial 2.5-fold dilutions (from 10−1 to 10−6.5) of the A549 cell culture supernatant were prepared in DMEM and used to infect Vero E6 cells; each dilution was tested in four replicates. The plates were incubated for at least 96 h and observed to monitor the development of cytopathic effect (CPE) using an EVOS Floid microscope (Invitrogen). Viral titers, expressed as TCID50/mL, were calculated according to both Reed and Muench and Karber methods based on three or four replicates for dilution (Reed and Muench, 1938 ).
3
Real-Time Cell Death Monitoring
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Cells were plated at a density of 1 × 105 per well in Black/Clear 96-well Plates in OPTIMEM culture medium supplemented with PI dye (1 µg/ml) and infected/treated as mentioned in figure legends. Red fluorescence is measured in real-time using a Clariostar plate reader equipped with a 37°C cell incubator or using an EVOS Floid microscope (Invitrogen). Maximal cell death was determined with whole cell lysates from unstimulated cells incubated with 1% Triton X-100.
4
Cell Adhesion and Viability Evaluation
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Cells were directly seeded onto specimens' surface (3 mm diameter) at a defined density (2000 cells/sample), and after 4 h of allowing adhesion, 450 μl of culture media was added to each sample. Subsequently, they were cultivated for 24 and 48 h; at each time point, the viability of the cells were evaluated using metabolic activity using the resazurin metabolic assay as prior described; moreover, the fluorescent Live/Dead assay was applied to visually check for viable cells (Live/Dead, Viability/Cytotoxicity Kit for mammalian cells, Invitrogen) with a digital EVOS FLoid microscope (from Life Technologies). Finally, the morphology of cells was visually investigated by FESEM imaging.
5
Cell Adhesion and Viability Evaluation
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Cells were directly seeded onto specimens' surface (4 × 4 mm2 square) at a defined density (2 × 104 cells/sample) and cultivated for 24 h allowing adhesion and spread. Then, the cells viability was evaluated by means of metabolic activity using the metabolic colorimetric alamar blue assay as prior described; then, to estimate the exact number of viable adhered cells, they were detached from specimens’ surface by trypsin, counted by the trypan blue using a Burker chamber and seeded into a new polystyrene plate. After 6 h adhesion into the new plate, the fluorescent Live/Dead assay was applied to visually check for viable cells (Live/Dead, Viability/Cytotoxicity Kit for mammalian cells, Invitrogen); images were collected with a digital EVOS FLoid microscope (from Life Technologies). Finally, the morphology of cells aligned following fibers orientation was visually investigated by FESEM imaging as prior detailed.
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