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Updated: Aug 9, 2026
Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches
Published on: November 21, 2018
Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging
M Bergeron1, J K A Nicholson, S Phaneuf
1National HIV Immunology Laboratory, Bureau of HIV/AIDS, TB and STD, CIDPC, PPHB, Health Canada, LCDC 0603B1, Tunney's Pasture, Ottawa, Ontario K1A 0L2, Canada.
Background:
In the past decade, human immunodeficiency virus (HIV) lymphocyte immunophenotyping has evolved significantly. New fluorochromes, new multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using the single-platform technique have all contributed to the reliability of T-cell subset measurements. In this study, four gating protocols were evaluated to select the most robust method for T-cell subset enumeration.
Methods:
Peripheral blood specimens from 21 HIV(+) and 20 HIV(-) individuals were monitored up to 96 h. Aliquots of specimens were stored at room temperature and analyzed at 6 (baseline), 48, 72, and 96 h. Aliquots were stained with CD45-fluorescein isothiocyanate (FITC)/CD3PC5/CD4RD1/CD8ECD. Data analysis was performed with all four gating protocols.
Results:
Only with fresh blood did all protocols provide similar results. From samples that were 48 h old, the choice of gating strategy had a dramatic impact on immunophenotyping results. The largest deviations from baseline values occurred at 96 h and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. The gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 h.
Conclusion:
By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days.
