[An improved colorimetric assay for chicken M-CSF like activity using chicken bone marrow cells cultured on micro-culture plates].
The role of M-CSF in the process of mammalian development has been drawing attention. Unlike mammalian embryonic development, in which M-CSF is supplied by the maternal body, avian embryonic development begins and proceeds under the influence of a certain amount of M-CSF present in each fertilized egg, and its concentration can be experimentally controlled while observing the embryo's developmental state. Apart from this fact, aves provide us with many other advantages in the study of embryonic development. It is, however, not easy to separate multipotent hematopoietic stem-cell fractions from avian bone marrow. In addition, because a macrophage colony forming assay in soft agar, though it has been widely used, requires a large number of cells and a long culture period, the method of assay is not adequate for the sensitive detection of M-CSF activity in small scale samples. We have established a method for the depletion of nucleated erythrocytes from chicken bone marrow cell suspensions using percoll density gradient centrifugation, and have also developed a new method for determining chicken M-CSF-like activity employing a liquid culture. In this method, a 100 microliters aliquot of fractionated hematopoietic stem cells, 1 x 10(5), was placed in a well of 96-well flat-bottom culture plate, 100 microliters of sample was then added to each well, and the uptake of neutral red was measured after 4 days of culture. These procedures represent a simple and sensitive means of detecting M-CSF-like activity in chicken serum of x60 to x32 dilutions.(ABSTRACT TRUNCATED AT 250 WORDS)
Shao
X
X
Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan.
Watari
E
E
Araki
T
T
Yokomuro
K
K
D000818:Animals; D001853:Bone Marrow / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002645:Chickens; D003124:Colorimetry; D003584:Cytological Techniques; D005260:Female; D016173:Macrophage Colony-Stimulating Factor / Q000032:analysis*; D016186:Receptor, Macrophage Colony-Stimulating Factor
D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor