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Application Notes

Robotic Microscopy with the Nikon Ti2 for high-content analysis applications

Robotic Microscopy—a combination of high-content screening methods—enables multivariate experimental approaches with large cell populations and member-level sensitivity. Here we explore how the new Nikon Ti2 line of inverted research microscopes is uniquely suited to Robotic Microscopy applications, focusing on work utilizing induced pluripotent stem cells (iPSCs) as disease models in drug screening.

Identification of astrocytes derived from neural progenitor cells by high-throughput screening using a live cell mRNA detection technology

Neural progenitor cells (NPCs) contain the capability to differentiate into neuronal and glial cell lineages, and have been used by researchers to develop ways to repair the central nervous system following damage.  One example is the derivation of astrocytes from NPCs, which is effective in generating functional terminally differentiated neuronal cells that can then be subjected to molecular and biological assays.  Currently, astrocytes are mainly identified using intracellular antibody staining of proteins such as GFAP, which prevents those characterized cells from use in further experiments, due to the need to permeabilize the cells for successful staining.

Here, we successfully demonstrate the detection of intracellular GFAP mRNA during the NPC-to-astrocyte differentiation process using a novel probe-based live-cell RNA detection technology, which can specifically measure RNA expression in whole cells without altering cell viability, function, or integrity.  This allows researchers to not only monitor the regulation of specific mRNA during the course of the differentiation protocol, but also enables interrogated cells to be used in downstream cell-based functional assays. Here, we employed a high-throughput screening (HTS) method to screen our NPC-derived astrocytes expressing GFAP mRNA, which retained their biological function as evident in a subsequent migration assay.   This enabled the quick and accurate identification of highest GFAP mRNA expressing cells, thus maximizing the likelihood of success in our downstream assay. 

The ability to monitor gene expression during NPC differentiation to astrocytes in a live cell setting not only removes the need to set up extra duplicate wells for downstream studies, but also enhances the relevance of the data generated, due to the fact that the same cell is being tracked throughout the entire experimental workflow.  Using live-cell RNA detection probes in HTS experiments not only enables researchers to vastly increase the number of cells that can be accurately interrogated at a time, but also allows them to retain their best candidate live samples for use in downstream functional assays, something that is not currently possible using traditional antibody-based methods.

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