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Development and Optimization of a High-Throughput Assay for Quantifying Phagocytosis in a Neutrophil-Like Cell Line
Citation:
Barbo, N., S. Martos, D. Phelps, J. Harrill, C. Willis, AND K. Slentz-Kesler. Development and Optimization of a High-Throughput Assay for Quantifying Phagocytosis in a Neutrophil-Like Cell Line. Carolinas SETAC 2024 Annual Meeting, Boone, NC, April 14 - 16, 2024. https://doi.org/10.23645/epacomptox.25664961
Impact/Purpose:
Presentation to Carolinas SETAC 2024 Annual Meeting
Description:
Phagocytosis is essential for proper innate immune defense across many organisms. When exposed to immunosuppressive environmental chemicals, phagocytosis may be inhibited, potentially increasing susceptibility to pathogens. To quantify the immunosuppressive potential of a chemical library, a high-throughput assay is necessary that assesses phagocytic function. We have developed a high-content imaging phagocytosis assay on the Opera Phenix+ using neutrophil-like HL-60 cells and fluorescent pH-sensitive E. coli-derived particles (pHrodo™ BioParticles™; Invitrogen). This assay allows high-throughput quantification of phagocytosis in neutrophil-like HL-60 cells following their exposure to chemicals. To test xenobiotic-induced changes in phagocytosis, five chemicals suspected of phagocytic inhibition were selected for initial testing: wortmannin (0.01 μM), SB 203580 (10 μM), perfluorohexanoic acid (PFHxA; 100 μM), phenanthrene (150 μM), and bisindolylmaleimide I (10 μM). Cytochalasin D (10 μM) was used as a positive control and D-mannitol (10 μM) was selected as an expected negative control. Neutrophil-like HL-60 cells were pre-treated with chemical for 30 minutes. Following this exposure, opsonized red pHrodo BioParticles were incubated with cells for 2 hours. pHrodo BioParticle fluorescence was then quantified within the cells to assess phagocytosis. As expected, the positive control, cytochalasin D, inhibited phagocytosis and the negative control, D-mannitol, did not inhibit phagocytosis. When compared to dimethyl sulfoxide (vehicle) treated control cells, phagocytosis was significantly decreased following exposure to PFHxA (66.0%), phenanthrene (83.9%) and SB 203580 (92.4%). Bisindolylmaleimide I and wortmannin did not significantly alter phagocytosis. Decreases in phagocytic function may weaken an organisms’ protection from pathogens. Therefore, identifying chemicals that can inhibit phagocytosis is important for assessing their potential immunotoxicity to humans and other species. The development of this in vitro, high-throughput assay will facilitate the screening of many more environmental chemicals, including PFAS, for their immunosuppressive potential via neutrophil phagocytosis inhibition. This abstract does not reflect the official policy of the US EPA.
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DOI: Development and Optimization of a High-Throughput Assay for Quantifying Phagocytosis in a Neutrophil-Like Cell Line
NBARBO_2024CSETAC_POSTER_FINAL.PDF (PDF, NA pp, 935.475 KB, about PDF)