Interrogating Cell Signaling

Biochemical Scale – ELISA: Get a bigger picture by combining stimulus readout with total protein

The Enzyme-Linked Immunosorbent Assay (ELISA) is a mainstay of many research labs that, when carried out carefully, provides powerful insight to biological processes. The assay is so well-established these days that ready-to-use kits are available that efficiently streamline experimental workflows. To quantitatively evaluate TGF-β signaling using SMAD2/3 phosphorylation as a readout, two ELISA kits were used: one that only detects phosphorylated SMAD2/3, and the other that detects total SMAD2/3. Measuring only phosphorylated SMAD2/3 can provide relative insight when comparing samples treated with various TGF-β concentrations, however by quantifying both phosphorylated and total SMAD2/3, those values can be further normalized via total protein load. The total SMAD2/3 values provide context as to the breadth of the stimulus response, which is insightful when comparing canonical and non-canonical signaling pathways.

Proteins tend to be labile when taken out of their native environment, such as when cells are lysed. Therefore, it is important to take great care to preserve them by including protein inhibitors in the cell lysis buffer, as well as quickly transitioning lysates to 4°C.

2D Cellular Scale - Nuclear Translocation: Chose an imaging modality and magnification that provides optimal statistical robustness

There are many variations on how to analyze and interpret nuclear translocation of a target molecule. The most straightforward method is to simply measure changes in nuclear signal. This can be easily done in Gen5 software by generating a primary mask using a nuclear marker, then quantifying target signal as a secondary mask. The power of high-throughput analysis lies in the ability to measure the nuclear signal of hundreds of thousands of cells across an entire microplate. Reliably accomplishing this for an object as large as a nucleus does not necessarily need an advanced imaging modality, such as confocal microscopy - widefield microscopy in most cases will almost always suffice. Additionally, the enhanced z-sectioning capabilities of confocal may introduce more complexity to samples that are not on the same z-plane by requiring z-stacks, which in turn may unnecessarily increase the overall data size of your sample set. In the same spirit of high throughput, lower magnification (10X or 20X) will provide higher n values for samples, leading to a higher degree of statistical certainty with a particular phenotype or response.

3D Multi-Cellular Scale – Spheroid: If possible, embed spheroids in a matrix and use an anti-fade reagent

Ultra-low attachment round-bottom microplates are great for forming spheroids and preparing them for imaging, however, due to the high diffraction of the curve surface, are very much not ideal for the actual imaging. It is recommended that spheroids are transferred to a flat-bottom vessel. In the associated application notes, an Agilent 96-well flat-bottom microplate was used (part number 204626-100). By the very nature of multi-well, high-throughput imaging, a microplate will experience movement to some degree which risks disturbing the location and orientation of the spheroid. Embedding spheroids in a matrix of some sort (2 mg/mL collagen was used, but agarose would suffice) will ensure that the spheroid will maintain its location and orientation.

High-intensity light tends to generate free radicals like reactive oxygen species (ROS), which in turn can denature and destroy both protein-based (GFP, RFP, etc.) and small molecule (Alexa Fluor, etc.) fluorophores, an effect called photobleaching. This is especially exacerbated when acquiring confocal z-stacks of a thick sample, where the entire z-column of the sample is repeatedly exposed to high-intensity laser light. Anti-fade reagents such as propyl gallate act as antioxidants that react out ROS and in doing so, help preserve fluorophores. For aqueous samples, regardless of the medium (PBS, glycerol, etc.), including an anti-fade reagent will certainly ensure that high-quality images are obtained.