10 Tips for Successful High Content Screening (HCS) Assays

The term High content screening (HCS) was first described in a paper by Giuliano et al. (1997), as a platform that allows high-throughput screening (HTS) of cells in an array at subcellular resolution using multi-coloured, fluorescence-based reagents for both specificity and sensitivity. High content screening is a combination of modern cell biology, automated high-resolution microscopy, flow cytometry and robotic handling and has therefore emerged as a compound testing platform for “phenotypic” cell-based assays. In HCS, vast quantities of biological data are collected and analyzed. In essence, it captures a compound’s molecular and phenotypic effects on a cell through a wealth of extracted image data. In contrast to traditional HTS, which has a single read out of activity, HCS allows measurements of many properties of individual cell simultaneously. It is this feature of multiplex readout, which gives HCS tremendous power and its utility. With this in mind, we are happy to provide simple but useful tips for improving daily tasks as well as the overall quality of your research. We have, therefore, compiled a list of ten tips for achieving high quality data by HCS.

1. Experimental design for HCS

Whenever possible, positive and negative controls should be set up in every assay. Positive controls exhibit desired response for the validity of the assay and serve as a comparison to identified hits. Negative controls usually produce no response and serve as the baseline or background. In some instances, a positive control may not be readily available. Once a condition has been identified that induces a measurable phenotypic change reproducibly, then it can serve as a positive control. Ideally, a positive control is of the same type as the reagent to be screened. For compound screening, the same compound at different concentrations could lead to different phenotypes due to the compound’s different potencies on different pathways. Therefore, it is essential to test compounds in a broad dose response concentration range to find all the phenotypes associated with the assay.

2. Cell lines

The easiest cellular models are immortalized and cancer cell lines. Not all derivatives of a given cell type retain properties. For example, some breast cancer cell lines have lost estrogen signaling. It is recommended to verify that the cell lines are functional using known reference compounds during assay development to ensure desirable pathways are performing well in the chosen cell lines. Cell lines can be mislabeled, contaminated or mishandled, making them inappropriate for the planned experiments. Examination of the cellular properties is essential. It is worthwhile to validate cell lines by genotyping.
A good understanding of proper handling of cell lines by managing growth rate characteristics and cell passage number limitation is recommended. The source of the cell line should have documentation along with information on phenotype and genotype. Fingerprint new cell lines as soon as they arrive in the lab and periodically thereafter. Fortunately, the cost of validating cell lines is falling thanks to improvements in testing techniques. One method, using short tandem repeat (STR) analysis to identify DNA sequences unique to a cell line, is now widely available, rapid and inexpensive. There are also online databases that allow STR fingerprints to be compared to verify cell line identity. For example, ATCC has an STR database of all of its human cell lines.

3. Replicates

The cost of replicates should be weighed against the cost of cherry picking hits and performing a confirmation screen. HCS assays with complex phenotypes are often difficult to score. Experiments are normally performed in duplicates or higher replicate numbers in order to minimize false positives and false negatives. Increasing the replicate number from 2 to 3 is a 50% increase in reagent cost. HCS screens are normally performed by first screening at a single concentration in duplicates and then retesting hits in confirmation assays.

4. Minimizing cross-talk due to bleed-through

An important consideration for HCS is that the fluorescent dyes used in assays typically have broad excitation and emission spectra. As a result, there can be significant bleed through from one fluorescent probe to another. To minimize this, wavelengths chosen should take into account the peak properties of the fluorescent targets to minimize cross excitation. The filters in the emission path must be optimized to minimize cross talk between the different fluorescence emitters. It is recommended to review the specifications of the filters to understand how they perform.

5. Plate selection

The type of plate chosen is critical for successful screening. Polystyrene-based materials are cost-effective and most cell types can attach without basement substrate materials or coating. Glass and quartz offer the best optical materials but are expensive and are used when optics and flatness of the plate is required to detect subcellular structures.
For plates, typically only the first and the last columns of multi-well plate are used for controls. Ideally, the treated samples should be in the middle wells. Unfortunately, this practice can render the assay susceptible to the well-known problem of plate-based edge effects, which lead to over- or under-estimation of cellular responses when normalizing by the control wells. Solid black polystyrene microplates reduce well-to-well cross-talk and background signal for fluorescent assay. Use of solid white plates is recommended to enhance readouts in luminescent assays.

6. Consistency and reproducibility of cellular models

Once an assay is developed, it requires optimization to large scale screening. It is recommended for initial pilot tests to be run in small scale to determine whether the assay is sufficiently feasible and reliable for HCS. Optimize the workflow and assess all steps to minimize waste and rework. Evaluate in advance potential issues related to reproducibility of the experimentation and the quality of the data. When long incubation time is required, significant edge effects are likely to appear. Suggestions to minimizing edge effects are described in our list of 10 Tips for Successful Cell Based Assays.

7. Calibration of liquid handling and dispensing systems

High content screening employs automated robotic platform and integrated peripherals for plate handling, cell incubators, liquid dispensing, and detection systems. Several time-consuming and repetitive steps are automated enabling a higher throughput of ~20,000 wells screened per day. Each of the liquid handling devices used in automation process requires validation for accuracy and reproducibility. Before setting up a protocol, thorough understanding of the operation and precision of the automatic liquid handling system is necessary. Regular calibration programs and verification checks help to reduce liquid handling quality error. Setting a default standard pipetting protocols is also recommended for initial standardization of an assay (Taylor et al., 2002).

8. Assay quality and acceptance criteria for HCS

Assay quality is typically determined according to the Z' factor (Zhang et al., 1999). This is a statistical parameter that in addition to considering the signal window in the assay also considers the variance around both the high and low signals in the assay. The Z’ factor has become the industry standard means of measuring assay quality on a plate basis. The Z’ factor has a range of 0 to 1; an assay with a Z’ factor greater than 0.4 is considered appropriately robust for compound screening although many groups prefer to work with assays with a Z’ factor greater than 0.6.
In addition to the Z’ factor, assay quality is also monitored through the inclusion of pharmacological controls within each assay. A Z’ factor of 0.5 within a validation day and across multiple days would indicate high quality assay. Assays are deemed acceptable if the pharmacology of the standard compound(s) falls within predefined limits. Replicate reproducibility could be used as a metric indicating assay quality.

9. Normalization of HCS data

Normalization is necessary to address systematic variations inherent in test conditions such as reagent aging or changes to compound concentrations due to evaporation. Data obtained with poorly calibrated apparatus could also differ from one side of the plate to the other. System errors are measurable and can be corrected with suitable statistical tools.

10. Assay response

It is important to determine the response for the assay by performing a time-course experiment. The time-course depends on the type of assay chosen. For example, calcium flux assays must be performed in live cells and measured within seconds. Some compounds can be added simultaneously while others would require pre-incubation. Factor in timing of addition when protocols are adapted for automation and liquid handling devices. Finally, determine the tolerance of the cells to chemical compounds such as DMSO or water used to solubilize compounds. DMSO is the most common solvent used in biological drug discovery and many compound libraries are delivered in DMSO.

From SCREEN-WELL® compound libraries to complex CELLESTIAL® fluorescent probes and reporter assays for monitoring autophagy, cell signaling and cytotoxicity, Enzo Life Sciences offers a comprehensive portfolio for high content screening, some of which are described below.

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