TY - JOUR
T1 - Enrichment of dog leukocyte subpopulations using density gradients
AU - Austin, D. R.
AU - Chan, S. C.
AU - Malley, A.
AU - Hanifin, J. M.
AU - Hirshman, C. A.
PY - 1985
Y1 - 1985
N2 - 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.
AB - 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.
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M3 - Article
C2 - 2995677
AN - SCOPUS:0021854699
SN - 0141-2760
VL - 17
SP - 53
EP - 57
JO - Journal of Clinical and Laboratory Immunology
JF - Journal of Clinical and Laboratory Immunology
IS - 1
ER -