The use of Ficoll-Hypaque double density gradients in the separation of avian granulocytes from other cell types for the purpose of cell flow cytometric analysis.
Avian peripheral blood and embryonic spleen cells were prepared for cell flow cytometry. The Ortho Spectrum III was the flow cytometer used in these experiments. The major objectives were to identify the location of lymphocytes and granulocytes in the cytogram displayed by flow cytometry, to develop a technique which would allow the collection of granulocytes relatively free of other cell types and to characterize the cell cycle within these cell populations. The cytogram of fresh avian cells developed in the Ortho Spectrum III revealed three characteristic cell clusters. Peripheral blood or embryonic spleen cells were separated on a Ficoll-Hypaque double density gradients into two distinct layers and a pellet. Light microscopic examination revealed the top layer of cells to be primarily lymphocytes while the middle layer of cells was granulocytes. Presentation of the cells from these layers to the Ortho Spectrum III revealed that granulocytes made up Cluster 3 while lymphocytes were included in the other clusters. The Ortho Spectrum III was employed to determine the presence of G1 (pre-DNA synthesis), S (DNA synthesis), and G2/M (post-DNA synthesis and mitosis) phases of cells in Clusters 1 and 2 and Cluster 3. While all the cells from peripheral blood were in G1, the embryonic spleen revealed cells in G1, S and G2/M in both Clusters 1 and 2 and Cluster 3.
D000818:Animals; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002645:Chickens; D003973:Diatrizoate; D005362:Ficoll; D005434:Flow Cytometry; D006098:Granulocytes / Q000166:cytology; D008214:Lymphocytes / Q000166:cytology; D013154:Spleen / Q000166:cytology
D003973:Diatrizoate; D005362:Ficoll