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.
Highlights
Measures of respiratory burst and phagocytic cell activity are frequently utilized to assess cellular immune function in teleosts
The nitroblue tetrazolium (NBT), which is yellow in color, diffuses into the cells where it is reduced by reactive oxygen species, superoxide, to produce formazan crystals, which upon dissolution in potassium hydroxide (KOH) and dimethyl sulfoxide (DMSO) produce a blue color
This dramatic color change can be measured with a standard plate reader and because the absorbance of the solution is directly related to the amount of superoxide produced, absorbance can be used to estimate respiratory burst activity
Summary
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 platebased phagocytic cell activity assays for use with kidney cells from the fathead minnow (Pimephales promelas), an emerging immunotoxicity model. 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
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