To determine the number of inclusion forming units (IFU), confluent wild type MEFs, dgat1/2 double knock-out, DGAT1-complemented and control dgat1/2 cells monolayers were infected with C. trachomatis LGV-L2 434/Bu at an MOI ~0.5 as described above in two sets of 96 well plates (one for input and one for output IFUs) in triplicates. At 30 hpi, cells were either fixed with 100% methanol (EMD Millipore) for 10 min on ice (input plate) or lysed by hypotonic lysis with sterile water (output plate), followed by addition of 5xSPG to a final concentration of 1xSPG (200 μl per well) and stored at -80°C, as previously described [43 (link)]. For enumeration of output IFUs, confluent HeLa cell monolayers were infected by centrifugation at 3,000xg for 25 min at 10 °C (as described above) with serial dilutions of the lysates obtained from the output plates, incubated for 30 h and methanol-fixed. Inclusions from input and output plates were immunostained with polyclonal anti-LGV-L2 sera followed by Alexafluor-conjugated secondary antibodies as described previously [44 (link)]. Inclusions were counted using a Cellomics ArrayScan Vti HCS automated fluorescent imaging system (ThermoFisher).
Cellomics arrayscan vti hcs
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cellomics ArrayScan VTI HCS is a high-content screening (HCS) system designed to perform automated image acquisition and analysis of cell-based assays. The system utilizes fluorescence microscopy to capture images of cells in multiwell plates and provides software tools for quantitative analysis of cellular parameters.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5, by Leica (1 mentions)
Axio imager a2, by Zeiss (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fluozin 3 am, by Thermo Fisher Scientific (1 mentions)
Cellrox, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
7 protocols using cellomics arrayscan vti hcs
1
Quantifying Chlamydia Inclusion Forming Units
2
Evaluating MC3T3-E1 Cell Adhesion
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After 1, 2, and 3 days of cell inoculation, the MC3T3-E1 cells were fixed with 2.5% glutaraldehyde at pH = 7.4. During observation, the sample was washed with PBS three times for 5 min each, and the excess water was absorbed by the filter paper. After removal, the cells were inverted with 10 μL of DAPI and stained for 5 min. The adhesion of cells was observed with a laser scanning confocal microscope (LSCM) (TCSSP5, Leica Microsystems, Heerbrugg, Germany) or a positive fluorescence microscope (Axio Imager A2, ZEISS, Oberkochen, Germany). After 3 days of cell inoculation, the cells were fixed in 5% POM, and cell growth was observed using an automatic Cellomics Arrayscan (VTI-HCS, Thermo Scientific, Waltham, MA, USA).
3
Automated High-Content Imaging of Neurite Outgrowth
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The Cellomics ArrayScan VTI HCS reader high-content imaging system (Thermo Fisher Scientific, Waltham, MA) was used for automated image acquisition and morphometric analyses as previously described.46 (link) Image analysis was performed using the vHCS Scan software package with a manually optimized version of the Cellomics Neural Profiling Bioapplication for neurite outgrowth analysis. MAP2 protein-positive expression was analyzed in Target Activation Bioapplication. Output from high content image analysis included measurement of neurite outgrowth (neurites per neuron, neurite length per neuron). The single neurite length was determined by dividing the neurite length of the entire neuron by the number of neurites.
4
FLS Proliferation Assay with EdU
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Inoculated FLSs (1 × 105 cells/well) in a 24-well plate were cultured and transfected using the BeyoClick EdU-448 cell proliferation kit (Beyotime Biotechnology #C0071S) by following the manufacturer’s instructions. We incubated each well with 10 μM EdU at 37°C for 12 h. Hoechst 33,342 was used for nuclear staining, and Thermo Scientific Cellomics ArrayScan VTI HCS (Thermo Fisher Scientific, United States) was used for EdU imaging and analysis. The EdU incorporation rate was calculated based on the ratio of EdU-positive cells (green cells) to FLS-positive cells (blue cells) (Bao et al., 2020 (link)).
5
Cell Motility Assay Protocol
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Briefly, 5 × 103 cells were plated into each well of a 96‐well plate and incubated at 37°C. After 24 hours, the culture medium was replaced with serum‐free RPMI 1640 medium, and cells were cultured for an additional 24 hours. The cells were then washed twice with ice‐cold PBS and stained with Hoechst 33342 for 15 minutes in an incubator. The cells were subsequently washed twice with ice‐cold PBS, and culture medium was added to each well. Cell motility was detected with a Cellomics ArrayScan VTI HCS (Thermo Scientific, USA) according to the manufacturer's instructions (5 replicate wells per group).
6
Cell Motility Assay Using Cellomics ArrayScan
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Briefly, 5×103 cells were plated into each well of a 96-well plate and incubated at 37°C. After 24 hours, the culture medium was replaced with serum-free RPMI 1640 medium, and the cells were cultured for an additional 24 hours. The cells were then washed twice with ice-cold phosphate-buffered saline (PBS) and stained with Hoechst 33342 for 15 minutes in an incubator. The cells were subsequently washed twice with ice-cold PBS, and culture medium was added to each well. Cell motility was detected with a Cellomics ArrayScan VTI HCS (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions (five replicate wells per group).
7
Imaging and Quantification of Cellular Processes
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Images were acquired and analyzed on a Thermo Fisher Cellomics ArrayScan VTI HCS reader using iDEVTM software. The filter settings for each dye were as follows: excitation/emission, 494/516 nm for FluoZinTM3AM and Fluo4AM (Molecular Probes, Life Technologies); excitation/emission, 644/655 nm for CellROX (Molecular Probes, Life Technologies); and excitation/emission, 350/461 nm for Hoechst 33342 (Molecular Probes, Life Technologies). Each dye was loaded into live MIN6 β cells or dispersed mouse islet cells according to the recommendations of the manufacturer. For transmission electron microscopy images, MIN6 cells were transfected with either scrambled siRNA or targeted siRNA for ZIP6 and ZIP7 knockdown and fixed, and images were acquired as described previously (23 (link)). Granule number was quantified manually using ImageJ software (24 (link)). Total Internal Reflection Fluorescence Microscopy images were acquired with a Nikon TE2000U TIRF microscope at 5 Hz with a 100-ms exposure time. Insulin granule mobilization and exocytosis were analyzed by Matlab (Math Works), ImageJ (National Institutes of Health), and Igor Pro software. For a detailed analysis, refer to a previous publication (25 (link)).
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