This article supplements part 1 "Problems and Solutions", published in G.I.T. Laboratory Journal issue 3-4, 2011,
authored by Roger Northwood, Valentin Bruttel, Jonathon Orme, Peter Simpson and Anthony Davies.

In an attempt to implement miniaturization within our own HCS/A screening workflows, we have performed a pilot study to establish the feasibility of conducting cell based assays at nano liter volumes in a microarray format. After extensive evaluation of the state of the art methodologies currently employed with the cellular micro array field. We determined that sequentially adding cells and reagents onto a glass microscope substrate was by far the most practicable for our experimental needs.

The ultimate goal of this study is to replicate the assay preparation and screening workflow as outlined earlier into a miniaturized screen. Indeed it is the miniaturization of the assay preparation stage of the screening process that will yield the tangible benefits both in terms of cost savings and logistically. It should be pointed out that assay size reduction depends almost exclusively on the liquid handler used. In this study we are using acoustic dispending technologies, however, there are also other more conventional tip based non contact technologies that are also capable of delivering nano liter volumes of reagent to predefined areas on our slides.

Cell Based Assays on a Microscope Slide
As can be seen from figure 1 we have setup cellular arrays comprising of 384 individual treatments per microscope slide. This was achieved by spot-wise addition of Poly L lysine (PLL) which was found to significantly improve cell adherence within the spots), followed by cells and reagents. To achieve the high level of accuracy in both the volumes of liquid dispensed and the positional accuracy required for this task, we utilised a very precise non-contact dispensing technology (the Labcyte Echo and Equator), which allowed us to ensure that the PLL solution, cell suspension and drug solution were applied to exactly the same spatial location on the slide.

This method has allowed for the reproducible spotting of cells into defined and physically discrete colonies. This arraying method also permits the convenient staining, labelling and imaging of cells, in addition to this we were also able to use this technology to perform a high content assay.

Proof-of-principle Assays Using High Content Analysis
To demonstrate the potential of this miniaturized assay system for use with High Content Analysis we studied the dexamethasone-induced translocation of the glucocorticoid receptor (GR) from the cells cytoplasm to the nucleus. The glucocorticoid receptor (GR), which belongs to the superfamily of ligand-activated nuclear hormone receptors, is physiologically activated by cortisol and other glucocorticoids. Due to their lipophilic nature, these hormones can diffuse through the cell membrane and bind to the GR, which, in the cytosol, is part of a heteromeric complex with Heat Shock Proteins and Immunophilins (Pratt & Toft, 1997). In response to activation by ligand binding, the glucocorticoid receptor translocates from the cytoplasmic to the nuclear compartment (see figure 3), where it is involved in the regulation the expression of various target genes (Kumar & Thompson, 2005). For this study we monitored the translocation of GR by measuring the depletion of this molecule from the cytoplasm (see figure 3 and 4).

In esophageal adenocarcinoma derived SKGT4 cells seeded and maintained on the microarray, the GR receptor translocation in response to dexamethasone treatment. Demonstrating that this cell based assay system is truly amenable to miniaturization, further image analysis revealed the following data. Cells treated with dexamethasone had a 44% depletion in GR positive area in the cytosol. This indicates that GR translocation from cytosol to nucleus has indeed occurred in these cells. The p Value from this experiments was highly significant (p = 1.9 x 10^-13). Interestingly a comparison of experiments performed in the miniaturised format vs the standard 96 well format revealed an almost 2X improvement in assay performance as measured by GR depletion (results not shown).

As can be seen from the proof-of-principle experiments it is indeed feasible to perform high content screens at the nano scale. However as will be seen from the sample workflow figure 1, logistically this technique would be very difficult to scale up for large scale screening. The major issue associated with the proposed work flow above are the inherent problems associated with working with such small volumes of liquid and the tendency for these to evaporate within seconds after dispensing. To overcome this issue we have developed a novel environmental buffering technology that completely prevents evaporation (see figure 5).
As can be seen from figure 5 we have developed a miniaturized 96 well plate with wells of a diameter of 1 mm. This nano well plate is constructed from a special solid gel like material, that both provides structure and completely prevents evaporation. The droplet seen in figure 5 was introduced into the well using acoustic dispending technology and can be maintained intact within a nano well for periods exceeding 24 hours. Hence this technology now makes possible the screening of large numbers of compounds in cell based assays in volumes as small as 10 nl.