Purification and characterization of silkworm hemocytes by flow cytometry.
Hemocyte functions are well-investigated in the silkworm, Bombyx mori, however, detailed analysis of each hemocyte subset has been hampered by the lack of appropriate separation method. Here we use an array of flow cytometric analyses to characterize silkworm hemocytes with various molecular probes, such as propidium iodide, green fluorescence protein, monoclonal antibodies, and fluorescent lectins. Of these, separation using propidium iodide was the simplest and provided most reliable results for the isolation of the hemocyte subsets. cDNAs were then synthesized from these sorted populations and subset-specific gene expression was examined by RT-PCR. Granulocytes, plasmatocytes, and oenocytoids expressed different classes of immune genes, suggesting that they have multiple roles in silkworm immunity. In contrast, a contribution of spherulocytes to immunity was not documented in that they failed to express most of the genes. The functions of spherulocytes are thus likely to be distinct from those of the other three hemocyte subsets.
Nakahara
Yuichi
Y
National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
Shimura
Sachiko
S
Ueno
Chihiro
C
Kanamori
Yasushi
Y
Mita
Kazuei
K
Kiuchi
Makoto
M
Kamimura
Manabu
M
D000818:Animals; D000911:Antibodies, Monoclonal; D012831:Bombyx / Q000166:cytology / Q000235:genetics / Q000276:immunology*; D002469:Cell Separation; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006434:Hemocytes / Q000166:cytology* / Q000276:immunology*; D020411:Oligonucleotide Array Sequence Analysis; D037121:Plant Lectins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D037121:Plant Lectins