Imaging of cells was performed after 24 h and 72 h from seeding, both in bright field and fluorescence. In the latter case, cells were incubated with a 2 μM calcein AM (LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen Corporation) and 1 μM Hoechst (Thermo Fisher Scientific) solution in PBS (concentration 1 : 1000), to assess intracellular esterase activity/viability and to stain the cellular nuclei, respectively. The staining solution was added to each well, followed by incubation at 37 °C for 10 min. Images of the blue channel (nuclei, bandpass filter value equal to 461 nm) and green channel (cytosol of viable cells, bandpass filter value equal to 517 nm) were acquired at a magnification of 10× with an optical microscope (ECLIPSE Ti-E Inverted epifluorescence microscope, Nikon Instruments).
Eclipse ti e epifluorescence inverted microscope
Manufactured by Nikon
The Eclipse Ti-E is an epifluorescence inverted microscope manufactured by Nikon. It is designed for advanced live-cell imaging and high-resolution fluorescence microscopy. The Eclipse Ti-E features a stable and vibration-resistant stand, a high-precision motorized stage, and a range of illumination options, including LED and mercury lamp sources. It supports a variety of fluorescence imaging techniques, such as TIRF, FRAP, and FRET, making it suitable for a wide range of applications in cell biology, neuroscience, and other life science research fields.
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7 protocols using eclipse ti e epifluorescence inverted microscope
1
Fluorescence Microscopy for Viability
2
Cell Density Quantification by DAPI Staining
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After 24 h or 7 days of incubation (depending on the test), the medium was removed and samples were washed with PBS 1X. Fixation was performed with 4% paraformaldehyde in PBS for 15 min, then the fixed cells were washed three times with PBS 1X (5 min for each wash) and treated with 0.1% Triton X-100 in PBS for 15 min. After that, fixed cells were washed 2 times with PBS 1X (5 min each wash) and incubated (15 min) at room temperature with 4′,6-diamidino-2-phenylindole (DAPI) 1 µL/mL in PBS to stain nuclei. Finally, cells were washed with PBS 1X (5 min) and observed with an epifluorescence microscope (ECLIPSE Ti-E Inverted epifluorescence microscope, Nikon Instruments). Pictures were taken at 10X magnification using a DAPI filter (440 nm) acquired with a high-resolution device camera and processed by means of the dedicated NisElements software (Nikon).
The Fiji software41 (link) (an implementation of ImageJ by the U.S. National Institutes of Health) was used to quantify the cell density of each sample, by counting the number of DAPI-stained nuclei present in each micrograph of 5 different and arbitrary areas per sample. Data were reported as mean and standard error of the mean (SEM).
The Fiji software41 (link) (an implementation of ImageJ by the U.S. National Institutes of Health) was used to quantify the cell density of each sample, by counting the number of DAPI-stained nuclei present in each micrograph of 5 different and arbitrary areas per sample. Data were reported as mean and standard error of the mean (SEM).
3
Reactivation of Latent Viral Infections
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HFF cells were treated with ACV (Sigma-Aldrich; catalog number PHR1254-1G) for 24 h before infection with different viruses (according to the experimental conditions required) carrying different fluorescent proteins, including OK41 (mCherry and mTurq2) OK11 (mCherry), OK12 (EYFP), OK22 (mTurq2), OK29 (mCherry), and OK35 (mCherry). The infected cells were maintained in specific conditions, 37°C with ACV-containing medium for 7 days. Next, the ACV was removed from the medium, and the quiescently infected cells were maintained in regular medium for 5 to 7 days until reactivation was induced. Reactivation was induced by infecting those cells with HCMV or lentivirus expressing VP16 transfection. Three to 5 days postreactivation, the plates were scanned using Nikon Eclipse Ti-E epifluorescence inverted microscope, and plaques were counted according to the fluorescent protein expressed.
4
Quantifying HSV-1 Genome Expression
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To estimate the number of HSV-1 genomes expressed in each infected cell, we obtained images (as described above) using a Nikon Eclipse Ti-E epifluorescence inverted microscope. Each experimental condition (different cells, viruses, inhibitors, and MOI) was replicated in two wells, and the experiment was performed at least twice. From an individual well, five random areas were imaged. From each image, 100 cells were analyzed for their color content. To define the average number of incoming genomes being expressed, we used the mathematical equation for estimating the most likely average number of genomes expressed in each cell (λ) according to the number of one- (r1), two- (r2), or three-color (r3) cells out of the number of cells analyzed (n), as was previously developed (Kobiler et al., 2010 (link)):
The λ was calculated for each well individually (based on 500 cells) and for each condition the mean λ and standard deviations were calculated. To predict the significance of the difference between the condition a two tailed student T-test was performed.
The λ was calculated for each well individually (based on 500 cells) and for each condition the mean λ and standard deviations were calculated. To predict the significance of the difference between the condition a two tailed student T-test was performed.
5
Reactivation of Latent Viral Infections
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HFF cells were treated with ACV (Sigma-Aldrich; catalog number PHR1254-1G) for 24 h before infection with different viruses (according to the experimental conditions required) carrying different fluorescent proteins, including OK41 (mCherry and mTurq2) OK11 (mCherry), OK12 (EYFP), OK22 (mTurq2), OK29 (mCherry), and OK35 (mCherry). The infected cells were maintained in specific conditions, 37°C with ACV-containing medium for 7 days. Next, the ACV was removed from the medium, and the quiescently infected cells were maintained in regular medium for 5 to 7 days until reactivation was induced. Reactivation was induced by infecting those cells with HCMV or lentivirus expressing VP16 transfection. Three to 5 days postreactivation, the plates were scanned using Nikon Eclipse Ti-E epifluorescence inverted microscope, and plaques were counted according to the fluorescent protein expressed.
6
Quantifying HSV-1 Genome Expression
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To estimate the number of HSV-1 genomes expressed in each infected cell, either U2OS, HFF, or HeLa cells were infected with an even mixture of three isogenic florescent viruses, OK11, OK12, and OK22 (35 (link)), each expressing a unique fluorescent protein under the immediate early cytomegalovirus (CMV) promoter: mCherry, EYFP, and mTurq2, respectively, at an MOI of 20. At 6–8 hours post infection (HPI), the infected cells were imaged using a Nikon Eclipse Ti-E epifluorescence inverted microscope. The experiment was repeated three times. From an individual well, five random areas were imaged. Three images from each well were analyzed for the presence of the three colors, with at least 200 cells analyzed in each. To estimate the most likely average number of genomes expressed in each cell (λ) according to the number of one - (r1), two - (r2), or three-color (r3) cells out of the number of cells analyzed (n), we used the model we have previously developed (22 (link)):
7
Quantifying Viral Infection Dynamics
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Vero cells plated in eight well NuncTM Lab-TekTM II Chambered Coverglass were used. Infection with OK11 was carried out in the presence or absence of 1.32 μM Trichostatin A (TSA) as described above. Images were acquired using a Nikon Eclipse Ti-E epifluorescence inverted microscope every 10 min with DAPI and RFP fluorescence at 37°C in a 5% (vol/vol) CO2 enriched atmosphere using a Chamlide TC stage top incubator system (Live Cell Instrument). Two experiments were done with technical repeats (wells) per condition. From each well, five frames were taken and analyzed. First, using the Imaris 8.1 (Bitplane) image software we identified the individual cells according to the Hoechst DNA staining. The level of red fluorescence at each time point was measured for each identified cell by the software. We removed all cells in which fluorescence levels did not increase above 10% during the infection, as most of these cells were either dead or resistant to infection. From each well, at least 200 cells were analyzed. The average levels of fluorescence from all the cells analyzed per experiment were normalized, and the average and standard deviation between experiments are presented.
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