Use Cell-Based Assays to Detect Immunofluorescence in 384-Well Plates

Imaging In-cell Western Multiplex

Western blots, step aside...

Although Western blotting is an established technique for detecting a specific protein within a complex sample, it has several inherent shortcomings. These include a lengthy workflow, low throughput, and limitations in the number of replicates, leading many researchers to seek an alternative method. This is where quantitative immunofluorescence in tissue culture, also known as in-cell Western blotting (ICW), comes in. Despite not having quite such a catchy name as Western blotting, the ICW is a simple technique that offers many advantages and is fast becoming a mainstay of many laboratory workflows.

In-cell Western taken from Azure Sapphire Biomolecular Imager
Figure 1. HeLa cells were serially diluted and seeded into a 96-well plate, cultured, fixed and permeabilized, then probed for Tubulin with AzureSpectra 550 (520 channel, green), beta-Actin with AzureSpectra 800 (785 channel, blue) and RedDot™1 Nuclear Stain as a normalization control (785 channel, red).

How does quantitative immunofluorescence in tissue culture work?

A major difference between Western blotting and ICW lies in how samples are prepared. While Western blotting requires that proteins be extracted, separated on a gel and then transferred to a membrane for detection, ICW is a newer method that uses cells cultured in 96- or 384-well microplates as the assay input.

During a typical workflow, cells are fixed (and permeabilized where targets are intracellular) in the microplate wells before being immunostained using fluorescently-labeled antibodies. The microplates are then read using a laser-based scanner, such as the Azure Sapphire Biomolecular Imager.

What are the advantages of in-cell Westerns as quantitative immunofluorescence?

Quantitative immunofluorescence in tissue culture provides many advantages over traditional Western blotting. First, because samples are prepared in microplates, assay throughput is significantly increased. This enables screening of multiple treatments in parallel, using a broader range of dilutions, and allows for a high number of replicates to increase the statistical significance of results. Greater throughput also provides time and cost savings, which are enhanced by the lack of requirement for specialized gels.

Another important advantage of quantitative immunofluorescence in tissue culture is that it conserves antigens in their quaternary structure and cellular location. The latter is especially useful to study trafficking of transmembrane receptors – something that is impossible when cells must be lysed for Western blot analysis. Quantitative immunofluorescence in tissue culture also benefits from high intra- and inter-experiment reproducibility, unlike Western blotting that can vary considerably between gels and between experiments run on different days.

What can in-cell Westerns be used for?

Because ICW are an incredibly flexible technique, it can be adapted for many different applications. These include monitoring changes in protein expression following RNA interference (siRNA), or in response to different concentrations of a treatment, and evaluating the occurrence of post-translational modifications upon treating cells with a particular stimulus or inhibitor.

In-cell Western blotting can also be used to study protein movement. In addition to analyzing transmembrane receptor trafficking, it is possible to follow the binding of fluorescently-labeled ligands to cell-surface receptors and to monitor their subsequent uptake. Where quantitative immunofluorescence in tissue culture is adapted to a fluorescent immunofocus assay, it allows the infectivity of non-cytopathic viruses to be investigated.

How do I read my plates with in-cell Western blotting?

You will need a laser scanner to be able to read from the bottom of a microwell plate. This scanner is essential to measure the readout from quantitative immunofluorescence in tissue culture. Camera-based systems are unsuitable as they image the microwell plates from above, allowing efficient imaging of only the bottom of the wells in a vertical line to the camera, although it is possible to circumvent this issue by using multiwell chambers and imaging each chamber slide separately.

The Sapphire is the answer for quantitative immunofluorescence in tissue culture. A serial dilution of HeLa cells was seeded into a 96-well plate, cultured, fixed and permeabilized. Columns 1-3 were probed for beta-Actin using AzureSpectra 550 (green), columns 3-6 were probed for Tubulin using AzureSpectra 800 (blue), and the entire plate was stained with RedDot1 Nuclear Stain as a normalization control (red). The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software.

in-cell Western with Azure Sapphire laser scanner

The Sapphire offers several advantages over other commercially available laser scanners, such as the benefits of 10-micron resolution (the highest on the market),  with software that provides precise focal plane adjustment, as well as an automated Z-scanning function is used to find the focus of microplates of different brands.

It also enables true multiplexed detection by using the NIR 700 (685nm laser) and NIR 800 (784nm laser) channels for target detection, both of which produce low autofluorescence and high signal-to-background ratios, meaning they can detect even low-expressed targets – and the green channel (520nm laser) for normalization against DNA content or total protein, researchers can readily monitor two targets in parallel, using the third readout as a measure of target abundance relative to the number of cells.

Is there anything else I should consider for ICW?

While quantitative immunofluorescence in tissue culture is highly versatile, it does have a few limitations. The first of these is that the sample must be a homogeneous population of cells that can proliferate in a culture plate, ideally as a monolayer; tissue lysates cannot be used in this assay. Secondly, some Western blot-validated primary antibodies raised against lineal epitopes may not recognize their target during quantitative immunofluorescence in tissue culture; however, primary antibodies validated for immunofluorescence should work.

Finally, it is critical that antibody reagents do not demonstrate cross-reactivity in an initial Western blot experiment to determine specificity for each target. Although non-specific bands can clearly be seen on a Western blot, these will go unnoticed during quantitative immunofluorescence in tissue culture, making highly-specific antibody reagents essential to the production of meaningful results.

Additional Resources for In-cell Westerns: