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Development and Optimization of a High-Throughput Assay for Quantifying Phagocytosis in a Human 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 Human Neutrophil-Like Cell Line. SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25475947
Impact/Purpose:
Presentation/Poster presented to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity Search for CCTE records in EPA’s Science Inventory by typing in the title at this link.https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25
Description:
Background and Purpose: Phagocytosis, a central effector function of neutrophils, is essential for proper innate immune defense. When exposed to immunosuppressive pharmaceutical and environmental chemicals, neutrophil phagocytosis may be inhibited, potentially increasing susceptibility to infectious disease. However, neutrophil phagocytosis is often not assessed when determining a chemical’s safety due to previously low-throughput protocols. To quantify the immunosuppressive potential of a chemical library, a high-throughput assay is necessary that assesses neutrophil phagocytic function. We have developed and optimized a high content imaging phagocytosis assay on the Opera Phenix+ platform using differentiated neutrophil-like HL-60 cells and fluorescent pH-sensitive pHrodo™ BioParticles™ E. coli conjugates (Invitrogen; pHrodo bioparticles). pHrodo bioparticles fluoresce in an acidic pH. Since the phagosome is acidified after phagocytosis, fluorescence of the pHrodo bioparticles allows for the uptake of heat-killed bacteria to be measured as a functional readout for phagocytosis. This assay will ultimately allow for high-throughput quantification of phagocytosis in neutrophil-like HL-60 cells following their exposure to chemicals of interest. Methods: To optimize this assay, a series of preliminary experiments were performed. All-trans retinoic acid (ATRA) differentiated, dimethyl sulfoxide (DMSO) differentiated, and undifferentiated HL-60 cells were imaged on the Opera Phenix+ to determine levels of autofluorescence in green, red, and deep red fluorescent channels. These data were used to select a differentiation agent and fluorescent wavelength of the pHrodo bioparticles that would allow for the most accurate imaging of phagocytosis. Differentiated HL-60 cells were then imaged with either immunoglobulin G-opsonized or non-opsonized pHrodo bioparticles to determine if opsonization would aid the cells in phagocytosis, allowing for potentially greater sensitivity for detecting inhibition. To test xenobiotic-induced inhibition of 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 for inhibiting phagocytosis and D-mannitol (10 μM) was selected as an expected negative control. All concentrations of chemicals tested were non-cytotoxic to differentiated HL-60 cells as determined in parallel chemical exposures measuring CellTiter-Glo® Luminescent Cell Viability Assay (Promega) luminescence on the CLARIOstar Plus plate reader. HL-60 cells were differentiated for 5 days using 1.25% (v/v) DMSO. On day 5, cells were plated and pre-treated with chemical for 30 minutes. Opsonized red pHrodo bioparticles were added and incubated with cells for 2 hours. Plates were then imaged on the Opera Phenix+ and fluorescence was quantified within the cells to assess phagocytosis. Results: When compared to ATRA differentiated HL-60 cells, HL-60 cells differentiated with DMSO showed less autofluorescence across the red (1.27% change from background), green (1.28% change from background), and deep red (7.92% change from background) fluorescent channels. Therefore, we decided to use DMSO as the differentiation agent and to expose cells to the red pHrodo bioparticles in chemical treatment experiments. We also determined that opsonizing pHrodo bioparticles was advantageous for this assay because it increased their cellular uptake, resulting in 89% more red fluorescence in cells when pHrodo bioparticles were opsonized compared to when they were not opsonized. As expected, the positive control, cytochalasin D, inhibited phagocytosis (100% decrease from DMSO control cells) and the negative control, D-mannitol, did not inhibit phagocytosis. When compared to . . .
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DOI: Development and Optimization of a High-Throughput Assay for Quantifying Phagocytosis in a Human Neutrophil-Like Cell Line
NBARBO_2024SOT_POSTER_FINAL.PDF (PDF, NA pp, 1602.173 KB, about PDF)