Abstract
Merocytophagy (“mero”, Greek for partial; “cytophagy” for cell eating) is a process by which cells acquire microbes and cytosolic material through phagocytosis of a small portion of neighboring cells upon cell-cell contact. Cell-cell contact dependent transfer events can be assessed through co-incubation of differently labeled cells. With these assays, it is difficult to analyze the recipient cells by microscopy or bacterial burden within only recipient cells. Therefore, we established a synchronized transfer assay that allows for recipient cells to be isolated from donor cells following transfer events at a high purity. Here, we present this assay in context of bacterial infections and cytosolic cellular staining. With this protocol, mechanisms of cell-cell contact dependent transfer events and the events following merocytophagy can easily be investigated.
Keywords: Cell transfer, Bacteria transfer, Bone Marrow Derived Macrophages, Transfer assay, Merocytophagy
Background
Phagocytic cells can acquire infectious material when microbes are extracellular or directly from infected cells (Steele et al., 2016). This host mediated process allows for initiation of an effective cellular response to clear infection (Ahmed et al., 2008). However, this process can be exploited by bacterial pathogens to expand their replicative niche (Ireton, 2013; Steele et al., 2016). We previously found that cells can acquire microbes and cytosolic material from neighboring cells through phagocytosis of a small portion of the infected cell which does not kill the donor cell (Steele et al., 2016 and 2019). This process known as merocytophagy (“mero”, Greek for partial; “cytophagy” for cell eating) provides a mechanism for the intracellular bacteria to disseminate and sustain infection (Steele et al., 2016 and 2019).To understand the mechanism of merocytophagy, we established a synchronized transfer assay that can be used to analyze a purified population of recipient cells following transfer events. Previously described transfer assays allow for the quantification of transfer events, whereas this assay allows for the analysis of the engulfed material. Specifically, engulfed material following a transfer event can be analyzed using microscopy or microbial burdens. Herein, we describe a detailed protocol of the synchronized transfer assay to analyze transferred bacteria and cytosolic dye.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Microscopy results were collected using a Leica DM400 upright fluorescence microscope. For each sample, 50 images were taken at each desired time point per experiment. Images were analyzed using Leica LAS X software. The number of cells that acquired material from donor cells can be determined by counting the number of recipient cells that obtained either bacteria or CFSE (Figure 2 for reference). Transwells (or physical separation) can be used to determine the number of phagocytosis events of extracellular material (Steele et al., 2016 and 2019). Figure 2. Representative microscopy of CFSE transfer events. A. CFSE stained donor BMDMs. B. CTR stained recipient BMDM. C. CFSE transfer to CTR stained recipient BMDM.
Recipes
Acknowledgments
The authors would like to thank the National Institute of Allergy and Infectious Diseases (NIH) for funding (R01AI082870 and R56AI139476).
Competing interests
No competing interests declared.
Ethics
All animals were handled according to approved institutional animal care and use committee (IACUC) protocol #4946 at Washington State University.
References
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