Journal: PLoS ONE

Article Title: Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays

doi: 10.1371/journal.pone.0214878

Figure Lengend Snippet: The microfluidic system (A) allows sequential injections of samples into the flowcell. The fluorescence microscope in (B) captures CFP- and YFP-FRET images of the entire cell array that can be analysed to give average values for the YFP/CFP fluorescence ratio. (C) The spots of the array are composed of approximately 50 fluorescent cells, each transfected with a receptor-coding gene and a fluorescent calcium sensor coding gene. Both receptor and sensor are expressed in each fluorescent cell of an array spot (D). Each spot on the array expresses a different receptor type. When the receptor interacts with a ligand, the G α 16GUST44 signalling cascade is activated: the G α protein acts via Phospholipase C (PLC) to convert PIP 2 into second messenger IP 3 which can interact with IP 3 receptors (IP 3 R) on the Endoplasmic Reticulum (ER). The IP 3 R are calcium ion channels which, when activated, transiently release calcium into the cytoplasm. The Twitch2B calcium sensor, expressed in the cytoplasm, is a fluorescent FRET probe which, upon binding of calcium ions, changes conformation and thereby increase the FRET efficiency between the CFP and YFP fluorescent proteins. These FRET changes are captured by the camera as a ratio change of CFP and YFP intensities.

Article Snippet: All measurement series were performed using a 150 μ l flowcell and the flowcell holder (Micronit Microfluidics B.V., Fluidic Connect PRO Chip Holder.)

Techniques: Fluorescence, Microscopy, Transfection, Binding Assay