A practical guide for assessing respiratory burst and phagocytic cell activity in the fathead minnow, an emerging model for immunotoxicity

Abstract

Measures of respiratory burst and phagocytic cell activity are frequently utilized to assess cellular immune function in teleosts. Respiratory burst predominately occurs in neutrophils and causes the release of reactive oxygen species to kill pathogens. Phagocytosis is the process by which pathogens are engulfed and destroyed by various immune cells. Though a variety of approaches have been utilized to measure respiratory burst and phagocytic cell activity, assays that rely only on common laboratory equipment (e.g., plate reader) may offer advantages over those that rely on more specialized equipment (e.g., flow cytometer). The goal of the current study was to optimize and validate the use of a colorimetric plate-based respiratory burst and fluorometric plate-based phagocytic cell activity assays for use with kidney cells from the fathead minnow ( Pimephales promelas ), an emerging immunotoxicity model. In addition, a protocol for the dissection of kidney tissue followed by the extraction of kidney cells, as well as recommendations and resources for future experiments utilizing each of these assays, are provided. All methods are optimized for use with the fathead minnow or similar teleost species. Respiratory burst and phagocytic cell activity are measured using a standard plate reader.

Study of Macrophage Activity in Cats with FIP and Naturally FCoV-Shedding Healthy Cats

“…A volume of 50 µL of medium was removed from the top of each well. Cells should have settled to the bottom and therefore not be removed from the plate, as reported by Hampton et al [ 21 ]. After vigorously vortexing, 5 µL of nitroblue tetrazolium (NBT) and 0.5 µL of phorbol 12-myristate 13-acetate (PMA) were added in triplicate to each sample and then the plate was returned immediately to the incubator for 1 h. Following incubation, 100 µL 70% methanol (MeOH) was added to each well and washed two more times.…”

Developmental thyroid disruption causes long-term impacts on immune cell function and transcriptional responses to pathogen in a small fish model

“…Respiratory burst and phagocytic cell activity were selected for the assessment of immune cell function given their importance to non-specific, innate immunity. Both assays were performed on cells harvested from the kidney according to methods outlined by Thornton Hampton et al 27 . Respiratory burst was determined using a nitroblue tetrazolium reduction assay whereas phagocytic cell activity was assessed via the engulfment of fluorescein (FITC)-conjugated Escherichia coli K-12 BioParticles (Thermo Fisher Scientific, Waltham, MA) (n = 5/group).…”