Assessing Viral Titer and Infection Efficacy in 12 Minutes

This application note introduces a swift and precise workflow for assessing viral transduction efficiency in vitro using the Araceli Endeavor® imaging system and Clairvoyance™ analysis software. By leveraging these advanced systems, researchers can rapidly quantify fluorescent protein expression, capturing the full spectrum of cellular responses to viral infection within a mere 12 minutes. The combined capabilities of high-resolution imaging and subcellular quantification without sacrificing speed or assay integrity, make it an ideal solution for high-content virology assays and other fluorescence-based screens. This method ensures actionable insights with unprecedented speed, facilitating quick iterations and robust experimental outcomes.

Assessing the efficacy of viral infection using fluorescent tags is an important primary outcome in high content- based virology assays, e.g. in testing antivirals (Panchal et al 2010), and is essential to successful assay development. Here, we present a high speed workflow for assessing viral transduction efficiency in vitro using high content imaging and analysis. This application note quantitatively assesses BacMam baculovirus transduction of emerald green fluorescent protein (GFP) in mammalian cells, yielding plotted actionable data in 12 minutes. GFP fluorescence is used as a readout for transduction efficacy: the more viral particles inside the cell, the more copies of GFP integrate into the host cells’ genome, and the brighter that cell fluoresces in the green channel. Accurate quantitative assessment of fluorescence is key to this assay: measuring how much GFP fluoresces in each cell/well as well as how many cells are positive for GFP (transduction efficiency). Calculating fluorescence on a per cell basis is essential as cells often exhibit large variation in viral load within a single culture (Suomalainen and Greber 2021). Maximizing well coverage ensures the full range of cellular responses to the virus are encapsulated, and resolution is needed to see the response of individual cells with subcellular accuracy, but these attributes often come at the expense of speed. An end-to-end workflow without compromising speed, coverage or detail is presented here. Araceli Endeavor® images this 96-well viral assay in <7 minutes for 3 channels, with maximized well coverage and submicron resolution, then Clairvoyance™ quantifies these fluorescent data on a subcellular basis, yielding effective dose response curves in <6 minutes.

Endeavor imaging and Clairvoyance analysis deliver speed and scale to allow for quick iteration without compromising assay integrity. Here, cells are first identified and segmented using nuclear and cellular markers, and green fluorescence quantified. Well and treatment-level mean is reported and cellular heterogeneity interrogated, quantifying fluorescence in individual cells to differentiate high and low responders, detailing the level of viral titer is needed to get all cells in the culture infected at varying levels. The cellular positivity workflow presented here is broadly applicable to a variety of fluorescent based screens. Often the expression of a fluorophore above a threshold identifies cells of interest for further measurement of a primary outcome (e.g. looking at mitochondrial morphology after a CRISPR-mediated knockout) or directly assayed as hits in reporter-based screen, e.g. protein-protein interaction assays (Nierode et al 2016). Altogether, this highlights the usage of Endeavor and Clairvoyance for precise fluorescent quantification at speed, going from a plate in hand to relevant, actionable graphs in under 12 minutes total (Figure 1).

Assay: Human epithelial-like cells (U2OS) were seeded at 7,500 cells/well in a glass bottom 96-well microplate (Grenier 655891) and cultured overnight in standard conditions (complete media, 5% CO2, 37°C). Cells were then transduced with baculovirus containing an emGFP cassette designed for horizontal gene transfer in mammalian cells (BacMam 2.0 Transduction Control, Invitrogen B10383), using a two-fold dilution series from a multiplicity of infection (MOI) of 0.78 to 100, and incubated for 16 hours at 37°C. Cells were washed and fixed with 10% formalin for 15 minutes, washed, then incubated for 30 minutes with 1:100,000 dilution of HCS CellMask™ DeepRed (Invitrogen H32721) to stain whole cells and 1:500 dilution of Hoecsht-33342 to identify nuclei.

Imaging and analysis: In 6 minutes 40 seconds the 96-well plate was imaged on Araceli Endeavor® with 3 channels (blue, green, far red) with maximized well coverage and 0.27um/pixel digital resolution. Clairvoyance™ used for all analysis, with illumination correction using Endeavor reference data automatically applied. Initial analysis done in 5 minutes 19 seconds, with machine vision-based detection and segmentation of nuclei to measure GFP intensity then individual cell- level hit calling using a 35x background mean intensity threshold to identify cells with high levels of viral transduction. Experiment plate map with treatments and dosage were loaded into Clairvoyance, and dose response curves with assay statistics generated using the Clairvoyance ‘Analyze’ function. More complex analysis was later performed, taking 13 minutes 31 seconds to measure and automatically bin nuclei into low (>7x background) medium (>15x) and high (>35x) transduced categories, as well as segment and measure the whole cell by using the prior detected nuclei to seed the center, then using an intensity-based segmentation on the far-red cell mask to define cell borders. All analysis and graphing done within Clairvoyance, except for linear regression (GraphPad Prism 10).

Baculovirus transduction led to robust, dose dependent GFP expression in these human epithelial cells after 16 hours (Figure 1), imaged with submicron resolution and maximized well coverage in 6 minutes 40 seconds on Endeavor. Clairvoyance measured mean intensity for individual nuclei, as they had the brightest staining (1a), with fluorescent intensity and positivity calls on a per cell basis made in 5 minutes and 19 seconds. Cells positive for a minimum amount of GFP (mean fluorescence >7.5x background) followed a dose response curve, achieving a maximal response near 100% of cells expressing GFP at the top viral dose (1c) with EC50=20.5. Overall, GFP expression in the cell associated clearly with baculovirus titer for both nuclei and the cell body (Figure 2), though GFP sequestered preferentially in the nucleus. Fluorescence linearly correlated with viral titer (2a), with a linear regression R²=.997, demonstrating the accuracy of this end-to-end analysis without compromising speed.

Cell positivity calls using varying GFP thresholds were generated, as the level of viral infection desired is highly assay dependent. Dose response curves quantifying percent of nuclei meeting thresholds for a high response (35x background) showed a different dose response dynamic (Figure 3). Notably, only ~50% of cells at the highest viral titer were scored as ‘high responders’, but nearly all cells at that titer had some GFP expression (Figure 1c). This belies the heterogeneity of this viral transduction: even at an MOI of 6.25 a small (0.5%) but meaningful population of high responders exist, yet >75% of that population were negative for GFP. Measuring transduction at the level of the individual cell rather than bulk fluorescence proves here to be essential, describing the heterogeneity in this experiment and producing actionable insight.

This intensity-based cell identification within Clairvoyance™ allows measurements (e.g. cell morphology, nuclear intensity, spot count) to be made and reported just on a defined population. Identifying and measuring only these infected cells with subcellular resolution is key to HCS viral assays (Panchal et al 2010). The workflow here can be more broadly applied to analysis of cells in a mixed population such as coculture experiments where the cell type of interest is labeled with an antibody (e.g. neurons and astrocytes in Anderl et al 2009) or reporter assays where cells fluoresce only in certain conditions such as gene expression, CRISPR-mediated cleavage, or a given protein-protein interaction (Nierode et al 2016). Araceli Endeavor® and Clairvoyance’s 12 minute scan and analysis time here means these assays can be done with unprecedented speed and detail, increasing throughput without compromising quality.

Endeavor’s maximized well coverage paired with single cell-level quantification in Clairvoyance ensures that you’re seeing the vast majority of cells in the well with subcellular precision (Figure 1b). This means that your assay can capture the full range of heterogeneity within the well, measuring rare events with subcellular detail, not missing a minority population of high expressors that could sink your viral infectivity assay. From this analysis, immediately actionable insight can be gleaned, as all figures displayed were generated within Clairvoyance.

Heatmaps such as Figure 2b are generated on the fly during the protocol refinement stage of Clairvoyance, offering immediate visual feedback while dialing in the analysis parameters, while a dose response curve is only 3 clicks away after a plate map is loaded into Clairvoyance. This cuts down on your iteration timeline considerably: you can generate this dose response curve only 12 minutes after loading your plate into Endeavor, and immediately feed that forward into your next experiment.

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