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为了研究某些因子对巨核细胞(MK)倍体的影响,并有利于流式细胞仪检测,我们对MK液体培养法进行了摸索和改进,取得了较为满意的结果.体会如下:1.取成年SD大鼠股骨,用Catch缓冲液将骨髓冲出,吹打、洗涤,制成单细胞悬液.此步骤多数报道是采用肝素抗凝,并用低pH值的PBS或培养液冲洗细胞.但肝素本身对MK生长具有一定的作用,故可能影响研究结果,而单独采用PBS或培养液,无法取得均匀的单细胞悬液.2.上述的细胞悬液中加入以Tris和氯化铵为主要成分的溶液,以达到溶解细胞而便于MK收集的目的.3.将细胞悬液轻轻铺在Percoll液上(密度梯度依此为1.020、1.060、1.080),4℃下400g离心30min.MK在骨髓细胞中所占的比例很少,采用传统的淋巴分离液(密度为1.077)进行分离,会造成MK的丢失.据文献报道,
In order to study the effect of some factors on megakaryocyte (MK) ploidy and to facilitate the detection of flow cytometry, we explored and improved the MK liquid culture method and achieved satisfactory results. Adult SD rat femur, bone marrow with Catch buffer out, blow, wash, made single cell suspension in this step is reported in most cases is the use of heparin anticoagulant, and washed with low pH PBS or culture medium, but heparin Itself has a certain effect on the growth of MK, it may affect the results of the study, and alone with PBS or culture medium, unable to obtain a uniform single cell suspension.2 The above cell suspension was added to Tris and ammonium chloride as the main component Of the solution, in order to achieve the purpose of lysing the cells and facilitate the collection of MK .3 The cell suspension was lightly spread on Percoll liquid (density gradient accordingly 1.020,1.060,1.080), centrifuged at 400g for 30min at 4 ° C, Cells accounted for a small proportion of the use of traditional lymphatic drainage fluid (density 1.077) for separation, will result in the loss of MK.According to the literature,