Geissler D, Peschel C, Boyd J, Konwalinka G
Wien Klin Wochenschr. 1984 Jan 6;96(1):8-11.
The recently described micro-agar culture system for cloning erythropoietic progenitor cells was used to study the optimum conditions for the growth of CFU-M. In this system human mononuclear cells from normal human bone marrow were suspended in agar and incubated for 12 days. Various concentrations of phytohaemagglutinin lymphocyte conditioned medium (PHA-LCM) and prostaglandin E (PGE) were added to the liquid overlayer in the presence of 2-mercaptoethanol (2-ME) for the stimulation of CFU-M. Human AB serum was used instead of fetal calf serum (FCS) in all experiments. A sigmoidal dose-response curve, with a plateau at a concentration of 5 to 10%, was obtained by the addition of different concentrations of PHA-LCM in the presence of 10(-6)PG-E. Under optimal conditions (5% PHA-LCM, PGE 10(-6)M) a linear relation was obtained between the number of seeded cells and the megakaryocytic colonies formed. For routine morphological analysis the whole agar layer was stained using the Pappenheim method. For further characterization of CFU-M, an immunofluorescence test with rabbit antihuman factor VIII related antigen was performed on the whole agar layer.
最近描述的用于克隆红细胞生成祖细胞的微琼脂培养系统被用于研究巨核细胞集落形成单位（CFU-M）生长的最佳条件。在该系统中，将来自正常人骨髓的人单核细胞悬浮于琼脂中并孵育12天。在2-巯基乙醇（2-ME）存在的情况下，向液体覆盖层中加入不同浓度的植物血凝素淋巴细胞条件培养基（PHA-LCM）和前列腺素E（PGE）以刺激CFU-M。在所有实验中均使用人AB血清代替胎牛血清（FCS）。在10(-6)PG-E存在的情况下，通过加入不同浓度的PHA-LCM获得了一条S形剂量反应曲线，在浓度为5%至10%时出现平台期。在最佳条件下（5%PHA-LCM，PGE 10(-6)M），接种细胞数量与形成的巨核细胞集落之间呈现线性关系。对于常规形态学分析，使用帕彭海姆法对整个琼脂层进行染色。为了进一步鉴定CFU-M，对整个琼脂层进行了兔抗人因子VIII相关抗原的免疫荧光试验。
