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Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy
Published on: October 1, 2012
A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium
Jie Shen1, Changzu Cai2, Zhilong Yu3
11] Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing, 100871, China [2] College of Engineering, Peking University, Beijing, 100871, China [3] School of Life Sciences, Peking University, Beijing, 100871, China.
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.
Insights
This study demonstrates how Dickkopf-1 (DKK1) protects cells from anthrax toxin. A novel microfluidic assay quantifies DKK1
Area of Science:
- Cellular biology
- Toxicology
- Biotechnology
Background:
- Investigating secreted protein function in real-time is challenging.
- Anthrax toxin internalization, mediated by Dickkopf-1 (DKK1) and its receptor, leads to cell death.
Purpose of the Study:
- To develop a method for monitoring the dynamic interaction of anthrax toxin components in live cells.
- To quantify the role of DKK1 in anthrax toxin internalization and cell viability.
Main Methods:
- Utilized an integrated microfluidic device for real-time, controlled cell culture.
- Performed parallel cell viability assays with continuous perfusion of conditioned medium containing secreted proteins.
- Exposed DKK1-knocked-down cells to anthrax toxin and assessed rescue effects.
Main Results:
- Exogenous DKK1 rescued DKK1-knocked-down cells from anthrax toxin-induced sensitivity.
- The microfluidic assay precisely quantified the dynamic interactions between toxin and DKK1.
- Provided independent evidence for DKK1's function in anthrax toxin internalization.
Conclusions:
- The developed microfluidic system enables high-throughput analysis of secreted protein function in real-time.
- DKK1 plays a crucial protective role against anthrax toxin by modulating its internalization.
- This work offers new insights into the mechanisms of anthrax toxicity and potential therapeutic strategies.
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