Automating Cell Biology

Create Your Own HCS Assay Using Trusted Molecular Probes® Dyes

(See a complete list of products discussed in this article.)

High-content screening (HCS), or high-content imaging, is an automated imaging technique that enables simultaneous assessment of multiple cellular parameters, in turn enabling the effects of tens of thousands of compounds to be measured in cells by a single laboratory in a single day. The cost of high-content instruments has decreased rapidly in recent years, making HCS technology more accessible to academic laboratories. While we offer a broad assortment of kits designed specifically for HCS, many researchers have used our classic Molecular Probes® fluorescent reagents to develop and validate their own HCS assays. We highlight a selection of these probes that have been cited in recent publications, demonstrating their utility in HCS assays for both basic research and drug discovery applications.

What Makes a Dye Suitable for HCS?

Because HCS platforms are typically used for multiparametric analyses, one of the most desirable attributes of an HCS probe is a narrow emission spectrum. Many of our probes meet this criterion and are offered in a variety of emission colors, making multiplexing easier. Also important are both brightness and photostability, which improve image and data quality and thus facilitate the automated image analysis. For optimal image segmentation (demarcation of cells from background), it is critical that the staining be as accurate as possible. In addition, because most high-content work is performed with fixed cells, the HCS probe must be compatible with fixed-cell imaging. Many of our traditional probes satisfy all of these criteria and are thus well suited to a variety of HCS applications.

Alexa Fluor® Dyes: Essential Antibody Labels for HCS Applications

The Alexa Fluor® family of dyes is widely used by researchers as a component of “home brew” HCS assays. To visualize specific proteins as part of a single-parameter or multiplex HCS assay, we offer a number of labeling kits for the direct attachment of intensely fluorescent Alexa Fluor® dyes to less than 10 μg up to 1 mg of primary IgG antibody. One of the advantages of directly labeled antibodies is that you can use more than one same-species antibody in a single staining experiment.

Three recent studies illustrate the use of Alexa Fluor® dye–labeled antibodies in HCS applications across various research disciplines. In a cancer research study, Pedersen and coworkers fluorescently labeled EGFR antibodies with Alexa Fluor® 488 and Alexa Fluor® 647 dyes in an HCS assay to assess antibody internalization, degradation, and growth inhibition of cancer cell lines [1] (Figure 1). In a neurobiology application, Alexa Fluor® 488 dye was conjugated to secondary antibodies to visualize β-tubulin; the probe was used in the development of an automated imaging analysis platform for neurons, for which measurements of complex cell morphology changes are both time consuming and labor intensive [2]. In a stem cell study by Low and coworkers, Alexa Fluor® 488 dye–conjugated secondary antibodies were used in HCS assays to track the neural stem cell markers nestin and Sox2 [3].


Internalization of an anti-EGFR antibody
Figure 1. Internalization of an anti-EGFR antibody. (A) Confocal images (400x) of Alexa Fluor® 488 dye–labeled Sym004 and cetuximab (two anti-EGFR antibodies) in HN5 cells after 15 min and 4 hr of incubation. Cells were treated with the labeled antibodies, fixed, and stained with Hoechst 33342 and HCS CellMask™ Blue Stain. (B) Number of spots per cell at different time points after treatment of HN5 cells with 10 μg/mL of the indicated antibodies (points, mean; bars, SE; *, P < 0.05; **, P < 0.01; Student’s t test, Sym004 vs. cetuximab). Imaging was performed with the PerkinElmer Opera® LX high-content confocal imaging system. Images reproduced with permission from Mikkel Pedersen, Symphogen A/S, Lyngby, Denmark, and from the American Association for Cancer Research [1].


HCS Probes for the Cytoskeleton

In addition to acting as a cellular scaffold, the cytoskeleton plays a pivotal role in organelle transport, cell division, motility, and signaling, making it central in both normal and disease processes including cancer. Thus, changes in cytoskeletal morphology and dynamics are commonly measured parameters in HCS studies. Phalloidin conjugates are widely used in HCS imaging applications to selectively label F-actin in fixed cells (Figure 2). Recent studies illustrate the use of phalloidin conjugates to model stem cell lineage fates (Alexa Fluor® 488 phalloidin [4]) and responses to various cytoskeleton-disrupting drugs (Texas Red® phalloidin [5]).


Morphological changes and cytoskeletal rearrangements in HeLa cells after cytochalasin D treatment
Figure 2. Morphological changes and cytoskeletal rearrangements in HeLa cells after cytochalasin D treatment.
Cells were treated with 10 μM cytochalasin D for 3 hr, then stained with HCS CellMask™ Blue Stain, Alexa Fluor® 488 Phalloidin, and anti–α-tubulin followed by an Alexa Fluor® 647 secondary antibody. (A) Untreated cells. (B) Treated cells. (C) Comparison of cell size in untreated vs. treated cells.

HCS Probes for Organelles

For accurate labeling of organelles in HCS assays, the MitoTracker®, LysoTracker®, and ER-Tracker™ dyes and the fluorescent ceramide analogs can be used to label the mitochondria, lysosomes, ER, and Golgi apparatus, respectively (Figure 3) in either live- or fixed-cell applications. Peng and coworkers leveraged the accuracy of the MitoTracker® and LysoTracker® probes in training their algorithm to estimate fluorescent signal in different subcellular compartments [6]. This feat represents a major advance for studies of proteins that are localized to more than one compartment.

For imaging live cells using HCS platforms, we offer a wide selection of organelle-targeted CellLight® reagents. CellLight® reagents are prepackaged baculovirus particles encoding GFP or RFP fusions that localize to specific organelles or other subcellular structures. CellLight® reagents are also compatible with antibody-based detection protocols in cells treated with formaldehyde-based fixatives.


High-content imaging of mitochondria, lysosomes, ER, and Golgi apparatus
Figure 3. High-content imaging of mitochondria, lysosomes, ER, and Golgi apparatus.
Live HeLa cells were loaded with 1 µg/mL Hoechst 33342 to stain nuclei, and stained with (A) 100 nM MitoTracker® Red CMXRos, (B) 200 nM LysoTracker® Red DND-99, or (C) 10 µM ER-Tracker™ Red for 10 min at 37°C in DPBS (+ 2 mM CaCl2 and 1 mM MgCl2) or with (D) 10 µM BODIPY® TR Ceramide complexed to BSA for 30 min at 4°C in DPBS (+ 2 mM CaCl2 and 1 mM MgCl2). Following loading, samples were washed in DPBS, incubated for 15 min at 37°C in prewarmed MEM (containing 10% FBS), and imaged on a Thermo Scientific Cellomics® Arrayscan VTI with a 20x objective and a TRITC filter set.

Solutions Tailored for HCS

In addition to the selection of “home brew” reagents discussed in this article, many of our other probes and kits for cell function are compatible with HCS platforms, including our assays for apoptosis, autophagy, cell cycle, cytotoxicity, and intracellular trafficking.

We also offer a wide selection of validated assays and reagents tailored specifically for HCS platforms. For image analysis strategies based on nuclear segmentation, the HCS NuclearMask™ stains are available in three different colors and stain live or fixed cells; the HCS NuclearMask™ Red Stain is “tunable” for nuclear versus cytosolic intensity by optimizing the concentration. For image analysis strategies requiring whole-cell segmentation, the HCS CellMask™ stains label the entire cell (i.e., cytoplasm and nucleus) and are applied to cells immediately after fixation and permeabilization. Available in five different colors, the HCS CellMask™ stains are bright and photostable with narrow emission spectra.

Our HCS reagents and kits are packaged in automation-compatible formulations, validated on multiple imaging platforms, and compatible with multiplex applications. See all of our products for High Content Screening.


  1. Pedersen MW, Jacobsen HJ, Koefoed K et al. (2010) Cancer Res 70:588–597.
  2. Wang D, Lagerstrom R, Sun C et al. (2010) J Biomol Screen 15:1165–1170.
  3. Low J, Blosser W, Dowless M et al. (2012) J Biomol Screen 17:152–162.
  4. Treiser MD, Yang EH, Gordonov S et al. (2010) Proc Natl Acad Sci U S A 107:610–615.
  5. Ng AY, Rajapakse JC, Welsch RE et al. (2010) J Biomol Screen 15:858–868.
  6. Peng T, Bonamy GM, Glory-Afshar E et al. (2010) Proc Natl Acad Sci U S A 107:2944–2949.


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