Dooley Douglas C, Oppenlander Barbara K
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA.
Methods Mol Biol. 2004;263:201-18. doi: 10.1385/1-59259-773-4:201.
Methods are described for the characterization of CD34 antigen modulation and its relationship to cell proliferation in early human hematopoietic cells. Toward that end, quiescent primitive CD34+ and CD34NEG cells are purified from mobilized peripheral blood (MoPB). Unlike CD34NEG cells from other sources, those from MoPB grow readily in stroma-free culture, facilitating their analysis. Using a lineage-depleted, low-density mononuclear cell fraction, CD34NEGCD38NEGLINNEG and CD34+CD38NEGLINNEG cells are purified by cell sorting. Cells are cultured in serum-free medium supplemented with early acting cytokines. Up- and downmodulation of CD34 antigen can be observed within 40 h of incubation. Samples are removed for analysis of expression of CD34, CD38 and lineage-commitment antigens as well as for cell proliferation as determined by expression of Ki67 antigen and uptake of pyronin Y. This approach permits an assessment of changes in CD34 and CD38 antigen expression by primitive LINNEG cells as they are activated for growth or remain in a quiescent state.
本文描述了用于表征人早期造血细胞中CD34抗原调节及其与细胞增殖关系的方法。为此，从动员的外周血（MoPB）中纯化静止的原始CD34+和CD34NEG细胞。与其他来源的CD34NEG细胞不同，MoPB来源的CD34NEG细胞在无基质培养中易于生长，便于进行分析。使用谱系耗尽的低密度单核细胞组分，通过细胞分选纯化CD34NEGCD38NEGLINNEG和CD34+CD38NEGLINNEG细胞。细胞在补充有早期作用细胞因子的无血清培养基中培养。在孵育40小时内可观察到CD34抗原的上调和下调。取出样品用于分析CD34、CD38和谱系特异性抗原的表达，以及通过Ki67抗原表达和派洛宁Y摄取确定的细胞增殖情况。这种方法允许评估原始LINNEG细胞在被激活生长或保持静止状态时CD34和CD38抗原表达的变化。
