Abstract
It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization.
Highlights
A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium
Anthrax toxin consists of three proteins; protective antigen (PA) and two toxic factors: lethal factor (LF) and edema factor (EF)[3,4]
We have recently reported that DKK1 plays an important role in the internalization and lethality of anthrax toxin through the ternary structure of lipoprotein receptor-related protein 6 (LRP6)-DKK1-Kremen[214]
Summary
A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium. Microfluidic devices have been applied to study the cell responses to secreted factors in the culture medium through two major ways: 1) create a chemical gradient, stable or dynamic, of soluble molecules by micro-patterns or well-regulated flows on-chip26–28, 2) constrain the physical distribution of different types of cells and control the direct or indirect interactions between cells[29,30,31,32,33]. Neither of these approaches is cost-effective to supply fresh conditioned medium, nor can they avoid the direct attachment between different types of cells
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