Flow-cytometric studies of the phagocytic capacities of equine neutrophils.
Abstract: Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and unquenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds. Den fagocyterande förmågan hos hästens neutrofiler studerades med flödescytometri. Kinetiken för adhesion och upptag av jästceller undersöktes. Fagocytosförloppet slutfördes snabbare när serum-opsoniserade jästceller användes än när IgG-opsomgerade jästceller var målpartiklar. Släckning med trypanblått ger möjlighet att eliminera falskt positiva händelser, som beror på jästaggregat utanför neutrofilen. Släckningen orsakade en förlust av celler med neutrofilers ljus spridningsegenskaper, men detta påverkade inte de uppmätta frekvenserna av fagocylerande neutrofiler. Endast smärre skillnader i fagocytoskinetiken kunde ses när släckta och osläckta prover jämfördes. Det konkluderas att flödes-cytometri är väl lämpat för studier av ekvina neutrofilers fagocytos av jästceller. Metodens största fördel är att man snabbt kan analysera stora provserier, vilket möjliggör undersökningar av hela besättningar.
Publication Date: 1995-01-01 PubMed ID: 8669382PubMed Central: PMC8095419DOI: 10.1186/BF03547669Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study conducted a thorough examination of the methodological processes and efficiencies related to the use of flow cytometry in the assessment of equine neutrophils’ ability to engulf and digest yeast cells. The significant findings suggested that flow cytometry is a well-fitted tool for studying equine neutrophil phagocytosis of yeast cells, enabling the rapid processing of larger samples.
Methodology
- The phagocytic properties (ability to engulf and digest foreign substances) of equine neutrophils were investigated using flow cytometry – an analytical cell-biology technique.
- Test samples encompassed two distinct classes of opsonized yeast cells – serum-opsonized and IgG-opsonized. Opsonized means that the yeast cells were coated with a substance (serum or IgG) to stimulate phagocytosis.
Key Findings
- The study discovered that phagocytosis occurred more rapidly when serum-opsonized yeast cells were employed, as opposed to IgG-opsonized yeast cells.
- A dye known as trypan blue was effectively utilized to decrease the fluorescence of non-ingested yeast cells. This step is critical because it enables the differentiation between cells that have been ingested and those that are merely attached.
Implications of Trypan Blue Quenching
- While trypan blue quenching resulted in the loss of cells with light scattering properties typical of granulocytes, this didn’t influence the calculated proportions of truly phagocytic neutrophils.
- Aggregation of yeast cells affected the results but did not hinder the determination of actively phagocytic cell frequencies. Consequently, trypan blue quenching eliminates a false positive due to yeast cell aggregation.
Benefits and Applications
- Flow cytometry proves suitable for studies examining the phagocytosis of yeast cells by equine neutrophils. Its major advantage is rapid sample processing, which is beneficial for large scale studies or herd examination.
Cite This Article
APA
Johannisson A, Gröndahl G, Demmers S, Jensen-Waern M.
(1995).
Flow-cytometric studies of the phagocytic capacities of equine neutrophils.
Acta Vet Scand, 36(4), 553-562.
https://doi.org/10.1186/BF03547669 Publication
Researcher Affiliations
- Department of Pathology, Swedish University of Agricultural Sciences.
MeSH Terms
- Animals
- Cell Cycle
- Female
- Flow Cytometry / veterinary
- Horses / blood
- Neutrophils / physiology
- Phagocytosis
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Citations
This article has been cited 1 times.- Flaminio MJ, Rush BR, Davis EG, Hennessy K, Shuman W, Wilkerson MJ. Simultaneous flow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes.. Vet Res Commun 2002 Feb;26(2):85-92.
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