A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820)

Abstract

A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,30,5,50-tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content. Abstract A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3 0 ,5,5 0 -tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content.

Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing

“…Electronic micrographs reveal that in some fish species, different size granules are present in neutrophils, allowing for at least potential segregation between MPO and AP at a granular level [14,41]. Neutrophil degranulation in fish has been described before, supporting results of this study [17,22].…”

“…Anterior kidney tissue from 16 (8 fish per day, two separate days) individual fish of each species was aseptically collected and granulocyte enriched fraction was separated using a previously described technique [13,17]. Briefly, anterior kidneys were dissected and collected in Hank's balanced salt solution without Ca, Mg and phenol red (HBSS CMF , Mediatech e CellGro, AK, USA), homogenized in a 15 mL tissue grinder (Wheaton, USA) and pelleted for 15 min at 250 g. The cell pellet was resuspended in HBSS CMF and gently placed over separation medium with a specific gravity of 1.078 g mL À1 (Lymphocyte separation medium 1078, Mediatech e CellGro, AK, USA).…”

“…Briefly, anterior kidneys were dissected and collected in Hank's balanced salt solution without Ca, Mg and phenol red (HBSS CMF , Mediatech e CellGro, AK, USA), homogenized in a 15 mL tissue grinder (Wheaton, USA) and pelleted for 15 min at 250 g. The cell pellet was resuspended in HBSS CMF and gently placed over separation medium with a specific gravity of 1.078 g mL À1 (Lymphocyte separation medium 1078, Mediatech e CellGro, AK, USA). Gradients were centrifuged for 30 min at 400 g, the cells at the interface were removed, washed, resuspended in HBSS CMF and total leukocyte counts and viability (trypan blue dye exclusion) were determined using a Neubauer-ruled hemocytometer [13,17]. Cell suspensions were adjusted to a standard concentration of 2 Â 10 7 cells mL À1 and the granulocyte to nongranulocyte proportion was determined by differential leukocyte counts (minimum of 200 counted cells per slide) on Hemacolor (Harleco, EM Science, NJ, USA) and Myeloperoxidase (391A staining kit, Sigma) stained cytospin preparations of cell isolates.…”

“…Measuring exocytosis of myeloperoxidase from primary neutrophil granules in vitro is a direct, rapid and quantitative method to assess the degranulation process in neutrophils. The assay based on MPO e H 2 O 2 oxidation of 3,3 0 , 5,5 0 e tetramethylbenzidine (TMB) is considered the least toxic of the peroxidase sensitive substrates, and was successfully used in fish [17,21,22]. The absence of myeloperoxidase in bird and some fish species heterophils suggests use of alternative enzymatic assays to measure degranulation [23e25].…”

“…Plate was immediately read on microplate reader at 405 nm. Percent of release for MPO and leukocyte AP was calculated as described before [17].…”