Protocol for simultaneous detection of PRRS virus infection and dying cell populations by a flow cytometry double-staining approach

Figures

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  Figure 1. Microscopy pictures of healthy and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) infected MARC-145 cells Microscopic pictures of (A) Healthy MARC-145 cells and (B) PRRSV Infected MARC-145 cells. The microscopic pictures were taken by the Olympus IX70 Fluorescent Microscope by using 20 x 10 objective (200 x magnification). The scale is 50 μM.

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  Figure 2. Overview of flow cytometry double staining method for simultaneous detection of dying, infected, and both dying and infected Marc-145 cells Cells are seeded and infected with PRRSV (A). Following infection, cells are harvested and stained with fluorescent dyes: Far-red fluorescent reactive dye and JP-24 FITC-conjugated dye in a single tube per condition (B). The stained cells are used in flow cytometry to determine which cells of the population are dying, infected, and both dying and infected based on the fluorescent intensity of the two markers (C). The output is visualized as a scatter plot. Quadrants in the plot distinguish between the following populations: healthy cells (bottom left), infected cells (bottom right), dying cells (top left), and cells that are both infected and dying (top right) (D).

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  Figure 3. Detection of dying, infected, and both dying and infected Marc-145 cells The output is visualized as a scatter plot that distinguishes between the healthy cells (bottom left), infected cells (bottom right), dying cells (top left), and cells that are both infected and dying (top right) (A&B). The negative control scatter plot represents healthy, uninfected cells with minimal cell death and no infected and dying cells (C). The infected sample shows a large percentage of cells that are infected but not dying (52.3%, lower right), a portion of the cells that are both infected and dying (22.2%, upper right), some cells that are dying but not infected (12.1%, upper left), and a small percentage of uninfected, healthy cells (13.4%, lower left) (D). This layout effectively shows how different populations can be visualized upon infection and cell death, with the double staining method allowing for clear discrimination between these populations.

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  Figure 4. Flow cytometry results of double-staining for simultaneous detection of virus-infected and dying cells Expected results from the double-staining method in comparison to the single-staining method, highlighting distinct cellular populations based on viral infection and cell death. To gauge the effectiveness of the dual staining approach, we compared its performance with the conventional single staining methods. (A) Dying cells: Results for Far-red fluorescent dye single vs. double stain. (A) Infected cells: Results of FITC-conjugated JP-24 single vs. double stain. A comparison of the traditionally used single-staining methods and the double-staining method utilized for assessing cell death and infection revealed no significant difference in the rate of cell death (Figure 4A) and infection (Figure 4B) between the two staining approaches upon infection. Consequently, based on our findings, we concluded that the double-staining method can effectively replace the traditional single-staining method without compromising the accuracy or reliability of the obtained results. Error bars indicate standard deviation across experiments, and statistical significance was determined using…