Abstract
Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.
Highlights
Caspase inhibitor[27], annexin V28, or specific overall stains (e.g., ConA-Alexa Fluor 48829, FITC-dextran[10], and Cell Tracker Green[21])
The conventional viability assay employing PI and SYTO 9, a multi-step method[22], was transformed into a single-cell, one-step method resolved in real time with microbial cultivation occurring inside a microfluidic device
C. glutamicum growth was impaired by 10 μM PI
Summary
Caspase inhibitor[27], annexin V28, or specific overall stains (e.g., ConA-Alexa Fluor 48829, FITC-dextran[10], and Cell Tracker Green[21]). We present a dynamic and non-invasive cell viability staining method employing a low PI concentration in combination with non-toxic counterstains, provided continuously to bacteria or yeast in a microfluidic growth chamber array. Staining experiments utilized a microfluidic PDMS-glass device designed for single-cell studies of Corynebacterium glutamicum and Escherichia coli[31,32,33]. This cultivation approach, in combination with time-lapse fluorescence microscopy, confers spatiotemporal insight into the phenotypic variations and dynamics of cellular death at the single-cell level. The immediately apparent fluorescence responses of this novel and dynamic staining approach facilitate the elucidation of survival strategies in small cell populations, such as antibiotic tolerance, temporal resistance, resuscitation after membrane potential loss, or the formation of membrane permeability transition pores (MPTPs). The spatial resolution of intracellular fluorescence distribution enables the visualization of specific details regarding cell death, such as the partial death of cell poles after antibiotic contact
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