A rapid, direct assay to measure degranulation of bovine neutrophil primary granules

Quade, Mark J.; Roth, James A.

doi:10.1016/s0165-2427(97)00048-2

Cited by 764 publications

(356 citation statements)

References 13 publications

Supporting

Mentioning

349

Contrasting

Unclassified

Order By: Relevance

“…When media with Ca 2C was used, neutrophils readily responded to stimulants and the MPO exocytosis was detectable within 1e5 min. This finding is in agreement with studies conducted on human, bovine and seabream neutrophils [12,13,15,16,30]. Cytochalasin B has been reported to be necessary for primary granule exocytosis in bovine, human, and gilthead seabream neutrophils in response to stimulants [16,30,31].…”

Section: Discussionsupporting

confidence: 90%

“…This finding is in agreement with studies conducted on human, bovine and seabream neutrophils [12,13,15,16,30]. Cytochalasin B has been reported to be necessary for primary granule exocytosis in bovine, human, and gilthead seabream neutrophils in response to stimulants [16,30,31]. Neutrophils often have been pre-treated with cyto B before being exposed to stimulants, but no significant difference was reported when bovine and seabream neutrophils have been exposed to cyto B and stimulants at the same time [16,30].…”

Section: Discussionsupporting

confidence: 87%

“…The MPO assay was originally described to quantitate MPO in human neutrophils for the specific measurement of primary granule exocytosis [15,16]. Also, modifications of the assay have been reported for gilthead seabream (Sparus aurata L.) [30].…”

Section: Discussionmentioning

confidence: 99%

“…Cytochalasin B has been reported to be necessary for primary granule exocytosis in bovine, human, and gilthead seabream neutrophils in response to stimulants [16,30,31]. Neutrophils often have been pre-treated with cyto B before being exposed to stimulants, but no significant difference was reported when bovine and seabream neutrophils have been exposed to cyto B and stimulants at the same time [16,30]. The addition of cyto B alone stimulated 11% MPO release in bovine neutrophils [16], but in fathead minnow kidney neutrophils exposed only to cyto B alone, the mean percentage of MPO release was 1.1% (range 0e5%).…”

Section: Discussionmentioning

confidence: 99%

“…The assay based on MPOeH 2 O 2 oxidation of 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) is considered the least toxic of the peroxidase sensitive substrates [15e17]. The assay was adapted for use with fathead minnow (Pimephales promelas Rafinesque, 1820) kidney neutrophils using a microtitre plate method previously described for bovine and human neutrophils [15,16].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Palić

Andreasen

Menzel

et al. 2005

Fish & Shellfish Immunology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Palić

Andreasen

Menzel

et al. 2005

Fish & Shellfish Immunology

Self Cite

View full text Add to dashboard Cite

show abstract

The effects of two dietary synbiotics on growth performance, hematological parameters, and nonspecific immune responses in Japanese Eel

Olowe,

Hamidoghli,

Choi

et al. 2024

J Aqua Anim Hlth

View full text Add to dashboard Cite

Feed additives have attracted increased attention in aquaculture due to their ability to modulate fish gut microbiota, resulting in improved fish growth and immunity. This study assessed two synbiotics' effects in Japanese eels: Bacillus subtilis with mannooligosaccharide (MOS) and Enterococcus faecium with fructooligosaccharide (FOS). Six diets, including a control (CON), oxytetracycline (OTC), and four synbiotic diets ‐ B.subtilis at 1 × 106 and 107 CFU/g with 5 g/kg MOS (BS6MO and BS7MO) and E. faecium at 1 × 106 and 107 CFU/g with 5 g/kg FOS (EF6FO and EF7FO) ‐ were fed to triplicate groups of 20 fish averaging 6.00 ± 0.07g for eight weeks. Fish fed the BSMOS diets showed significantly higher weight gain (WG), specific growth rate (SGR), and feed efficiency (FE) compared to the CON and OTC diets (P < 0.05), but not significantly different from those fed the EFFOS diets. Weight gain, SGR of fish fed EFFOS were not significantly different from those fed all other diets (P > 0.05). Fish fed the OTC diet showed a higher mean aspartate aminotransferase (AST) level, though the difference was not statistically significant. The myeloperoxidase (MPO) activity of fish fed the BS7MO diet was significantly higher than in all other diets, and the superoxide dismutase (SOD) activity of fish fed the BS7MO diet was also significantly higher than in the EF7FO diet. Overall, the BSMOS synbiotic diets were significantly more effective than the CON diet in enhancing fish survival against Vibrio anguillarum. Our findings suggest that synbiotics can be a preferable alternative to antibiotics in aquaculture.

show abstract

Phagocytosis and peroxidase release by seabream (Sparus aurata L.) leucocytes in response to yeast cells

2003

View full text Add to dashboard Cite

A flow cytometric method was adapted to evaluate phagocytosis by gilthead seabream leucocytes after incubation with yeast cells (Saccharomyces cerevisiae). Head-kidney leucocytes were incubated in vitro for different times in different proportions with heat-killed fluorescein isothiocyanate (FITC)-labeled yeast cells to study the kinetics of phagocytosis. Attached and internalized yeast cells were differentiated by quenching FITC-labeled S. cerevisiae with trypan blue dye. Only internalized cells kept their FITC fluorescence after quenching. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity, as demonstrated by transmission electron microscopy (TEM). From the ultrastructural features of the phagocytic process, it was observed that cytoplasmic granule membranes fused with the phagocyte membrane at the point where the yeast cell was attached to the phagocyte surface. This observation led us to adapt a colorimetric method to study peroxidase (myeloperoxidase and eosinophil peroxidase) release, since both are considered to be markers of the degranulation that occurs in seabream head-kidney leucocytes in response to yeast cells. Head-kidney leucocytes were incubated with calcium ionophore (CaI), phorbol myristate acetate (PMA), or yeast cells for different periods of time (0 -30 min) to study the kinetics of peroxidase release. The results obtained indicate that CaI and yeast cells, but not PMA, stimulate the degranulation (by about 44.51% and 21.04%, respectively, at 30 min) of seabream headkidney leucocytes. Anat Rec Part A 272A: 415-423, 2003.

show abstract

A rapid, direct assay to measure degranulation of bovine neutrophil primary granules

Cited by 764 publications

References 13 publications

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

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

The effects of two dietary synbiotics on growth performance, hematological parameters, and nonspecific immune responses in Japanese Eel

Phagocytosis and peroxidase release by seabream (Sparus aurata L.) leucocytes in response to yeast cells

Contact Info

Product

Resources

About