RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector’s function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2021
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| Subjects: | |
| Online Access: | http://ir.unimas.my/id/eprint/36897/1/invasive1.pdf http://ir.unimas.my/id/eprint/36897/ https://www.sciencedirect.com/journal/analytical-biochemistry https://doi.org/10.1016/j.ab.2021.114432 |
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| Institution: | Universiti Malaysia Sarawak |
| Language: | English |
| Summary: | Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of
this vector’s function to obtain insights for improving its efficiency. Invasion is traditionally quantified by
antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from
infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires
supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces
its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based highthroughput
and inexpensive method for tracking bacterial adherence and internalization through flow cytometry
to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population
of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and celldependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host bacteria interaction. |
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