High content screening microscopy analysis

We present in this article a protocol to perform parallel analysis of hundreds of different genes on a single Lab-Tek chamber. We focused on a transfection protocol, as it has been apphed successfully in our laboratory in combination with high content screening microscopy analysis. It may serve as a basis for any subsequent adaptations of the method to more complicated analysis methods or cell systems. 

Analysis of the Samples by High Content Screening Microscopy... 

In this chapter we describe protocols for reverse transfection to generate mammalian cell arrays for systematic gene knock-downs by RNAi or knock-ins by ectopic cDNA expression. The method is suitable for high content screening microscopy at a high spatial and temporal resolution allowing even time-lapse analysis of hundreds of samples in parallel. 

The method comprises five individual steps (see Fig. 1), including the preparation of the transfection solutions, followed by their spotting onto a cell substrate (e.g., Lab-Tek culture dishes, Nalge Nunc International, Rochester, NY), plating of the cells onto the arrays of spotted transfection solutions, preparation of the transfected cells for functional analysis, and finally the analysis of transfected cells by high content screening microscopy. 

Analysis of samples by high content screening microscopy... 

Fig. 1. The five steps to produce arrays of transfected mammalian cells for high content screening microscopy. 1. Preparation of the transfection solutions on an automated liquid handler. 2. Spotting of the transfection solutions with a spotting Robot, for example, ChipWriter Compact on a cell substrate, for example, Lab-Tek tissue culture dishes. 3. Plating of the cells on dishes with dried transfection solutions. 4. Preparation of samples for functional analysis, for example, immunostaining. 5. Analysis of samples by high content screening microscopy.

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