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Flow cytometric procedure to detect apoptosis of bovine polymorphonuclear leukocytes in blood
K Van Oostveldt1, H Dosogne, C Burvenich
1University of Ghent, Faculty of Veterinary Medicine, Department of Physiology, Biochemistry and Biometrics, Merelbeke, Belgium.
Abstract:
A flow cytometric technique was used to detect apoptosis and necrosis of bovine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothiocyanate labeled annexin-V and propidium iodide (PI). Isolation of PMN from the blood following lysis by water or NH4Cl resulted in false positive results for apoptosis. Therefore, a method was developed to identify living, apoptotic and necrotic PMN simultaneously in a single 100 microl blood sample. To establish a positive control for PMN apoptosis, the effect of cycloheximide, actinomycin D, diamide, buthionine sulfoximine and sodium arsenite, that have been described to induce apoptosis by various mechanisms was tested. Only actinomycin D induced a significant increase in the percentage of apoptotic PMN after 2 h. Incubation of blood for 6 h with cycloheximide, actinomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. Sodium arsenite, mainly caused necrosis after 6 h of incubation.
Insights
This study developed a new flow cytometry method to accurately detect bovine polymorphonuclear neutrophil leukocytes (PMN) apoptosis and necrosis. The optimized technique avoids false positives, enabling precise analysis of cell death in blood samples.
Area of Science:
- Veterinary Immunology
- Cell Biology
- Immunotoxicology
Background:
- Accurate detection of apoptosis and necrosis in bovine polymorphonuclear neutrophil leukocytes (PMN) is crucial for understanding immune responses.
- Previous flow cytometric methods using annexin-V and propidium iodide (PI) were confounded by false positive apoptosis results after PMN isolation via lysis.
- A reliable method is needed to simultaneously identify viable, apoptotic, and necrotic PMN in whole blood samples.
Purpose of the Study:
- To develop and validate a flow cytometric technique for the simultaneous detection of apoptosis and necrosis in bovine PMN.
- To establish reliable positive controls for inducing PMN apoptosis using various chemical agents.
- To optimize sample preparation to prevent artifactual induction of apoptosis during PMN isolation.
Main Methods:
- A flow cytometric assay using fluorescein isothiocyanate labeled annexin-V and propidium iodide (PI) was employed.
- A novel method was established to analyze living, apoptotic, and necrotic PMN directly from a 100 microl blood sample, avoiding cell isolation lysis steps.
- Bovine PMN apoptosis was induced and assessed using cycloheximide, actinomycin D, diamide, buthionine sulfoximine, and sodium arsenite as positive controls.
Main Results:
- Incubation of whole blood for 6 hours with actinomycin D, cycloheximide, and buthionine sulfoximine significantly increased the percentage of apoptotic PMN compared to controls.
- Actinomycin D induced a significant increase in apoptotic PMN after only 2 hours of incubation.
- Sodium arsenite primarily induced necrosis in PMN after 6 hours of incubation.
Conclusions:
- The developed flow cytometric technique accurately distinguishes between viable, apoptotic, and necrotic bovine PMN in whole blood.
- This method overcomes the limitations of previous techniques that yielded false positive apoptosis results due to cell lysis.
- The study provides a validated approach for assessing PMN cell death and evaluating the effects of various agents on bovine neutrophil apoptosis and necrosis.