Related Experiment Video
Updated: Aug 18, 2026
Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform
Published on: October 22, 2021
Flow cytometric method for enumeration and characterization of newly released polymorphonuclear leukocytes from the
Chih-Horng Shih1, Beth A Whalen, Yukinobu Goto
1James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital-University of British Columbia, Vancouver, BC, Canada V6Z 1Y6.
Abstract:
Inflammation accelerates polymorphonuclear leukocyte (PMN) release from the bone marrow, and these PMNs are implicated in inappropriate tissue injury. We have previously developed a method using 5'-bromo-2'-deoxyuridine (BrdU) to study PMN kinetics using an immunocytochemical grading system of PMN on cytospin slides. The aim of this study was to develop a flow cytometric method to quantify the number of positively stained PMN and grade the intensity of staining for the transit time calculation of PMN through the marrow. Dividing myeloid progenitors in the marrow of rabbits were labeled with a pulse dosage of intravenous BrdU. BrdU-labeled PMN (PMN(BrdU)) were detected in the circulation using a FITC-conjugated anti-BrdU monoclonal antibody. The PMN(BrdU) were assigned to five groups according to their FITC intensity, and the transit times of PMN at different stages of development in the marrow were calculated. Results were compared using parallel immunocytochemical analysis of the same samples. In control animals, PMN(BrdU) in the circulation peaked at 72 h after BrdU labeling with 36.0% of PMN labeled. In normal rabbits, the transit times of PMN through the mitotic pool (49.5 +/- 4.2 h) and maturation pool (65.5 +/- 3.1 h) correlated well with immunocytochemical analysis and previously published values. Using this method, we demonstrated that exposure to air pollution particles accelerates the release of PMN(BrdU) from the marrow. We conclude that a flow cytometric approach for identifying BrdU-labeled leukocytes provides an objective and accurate method for studying leukocyte kinetics and behavior.
Insights
This study introduces a flow cytometry method to track polymorphonuclear leukocytes (PMNs) using 5'-bromo-2'-deoxyuridine (BrdU) labeling. This technique accurately measures PMN transit times and reveals that air pollution accelerates PMN release from bone marrow.
Area of Science:
- Hematology
- Immunology
- Cell Biology
Background:
- Inflammation triggers accelerated release of polymorphonuclear leukocytes (PMNs) from bone marrow, contributing to tissue injury.
- Previous studies utilized 5 extcdot-bromo-2 extcdot-deoxyuridine (BrdU) and immunocytochemistry to assess PMN kinetics.
- A need existed for a more quantitative method to determine PMN transit times through the marrow.
Purpose of the Study:
- To develop and validate a flow cytometric method for quantifying BrdU-labeled PMNs (PMN(BrdU)).
- To calculate PMN transit times through bone marrow mitotic and maturation pools.
- To assess the impact of environmental factors, such as air pollution, on PMN kinetics.
Main Methods:
- Rabbits received intravenous BrdU to label dividing myeloid progenitors.
- BrdU-labeled PMNs in circulation were detected using FITC-conjugated anti-BrdU antibody.
- PMN(BrdU) were categorized by FITC intensity for transit time calculation, validated against immunocytochemistry.
Main Results:
- Flow cytometry accurately quantified PMN(BrdU) and their staining intensity.
- Normal rabbit PMN transit times were calculated for mitotic (49.5 ± 4.2 h) and maturation (65.5 ± 3.1 h) pools.
- Exposure to air pollution particles was shown to accelerate PMN release from bone marrow.
Conclusions:
- Flow cytometry offers an objective and precise method for analyzing leukocyte kinetics.
- This approach enables detailed study of PMN behavior and response to stimuli.
- The findings highlight the influence of environmental exposures on hematopoiesis and immune cell release.
More Related Videos
09:57Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia
Published on: March 5, 2018
06:33Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia
Published on: November 10, 2023