Enrichment of dog leukocyte subpopulations using density gradients

D. R. Austin, S. C. Chan, A. Malley, J. M. Hanifin, C. A. Hirshman

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Density gradient methods have been used for the enrichment of leukocyte subpopulations from human blood. We have adapted a three step method utilizing centrifugation on Hypaque-Ficoll (HF), double density HF (ddHF) and Percoll density gradients to separate lymphocytes, monocytes and basophils from dog blood. After HF separation, both Basenji-Greyhound (BG) and mongrel dogs had similar percentages of lymphocytes and monocytes, but BG dogs had significantly greater (p > 0.025) numbers of granulocytes. When cells from HF gradients were spun directly on Percoll, the presence of granulocytes decreased the purification of leukocyte subpopulations. An intervening separation on a ddHF gradient removed granulocytes without a selective loss of lymphocytes or monocytes. When cells from ddHF gradients were further separated on Percoll, 2 distinct cell layers resulted. Layer 1 was monocyte rich with 72.4 ± 6.5% monocytes, 26.6 ± 6.2% lymphocytes, and 0.8 ± 1.8% granulocytes. Layer 2 was lymphocyte rich with 74.3 ± 11.1% lymphocytes, 21.4 ± 7.8% monocytes and 3.9 ± 4.2% granulocytes. Viability as determined by Trypan blue exclusion was above 90%. Using cAMP-Phosphodiesterase (PDE) as a marker, higher PDE activity was present in the monocyte rich layer. This was similar to that reported in humans. Basophil numbers were enhanced by the use of a 1-step separation on a ddHF gradient. The percentage of basophils was increased 10-fold over baseline levels, with a viability of 90%. These techniques can be used to purify various canine leukocyte subpopulations and provide cells for biochemical analysis.

Original languageEnglish (US)
Pages (from-to)53-57
Number of pages5
JournalJournal of Clinical and Laboratory Immunology
Volume17
Issue number1
StatePublished - Oct 10 1985

ASJC Scopus subject areas

  • Immunology

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