Strelkauskas A J, Teodorescu M, Dray S
Clin Exp Immunol. 1975 Oct;22(1):62-71.
Rosette techniques are presented for the enumeration and separation of both Ig+ T- and Ig- T+ human lymphocytes. In order to enumerate Ig+ cells, the direct immunocytoadhesion technique was employed using human erythrocytes (E) coated with purified anti-kappa or anti-lambda light chain antibodies. Specificity of these rosettes was shown with chronic lymphocytic leukaemias of either the kappa or lambda type. T+ cells were enumerated by a new indirect rosette technique in which the lymphocytes were initially treated with rabbit anti-human thymus cell antiserum followed by direct rosetting with human E coated with purified anti-rabbit light chain antibody. For normal individuals, 24-32% Ig+ T- cells and 65-71% Ig- T+ cells were found among the lymphocytes of peripheral blood as well as tonsils with these rosette methods. The Ficoll-Hypaque method was used to obtain purified Ig- T+ and Ig+ T- cells by removing rosetted Ig+ cells or T+ cells, respectively. The purity of the Ig- T+ cells was indicated by greater than 99% indirect rosetting of cells sensitized with anti-human thymus cell antibody (Ab) and by less than 1% direct rosetting with anti-kappa Ab-E+ anti-lambda Ab-E. The purity of the Ig+ T- cells obtained was indicated by 92-96% direct rosetting with anti-kappa Ab-E+anti-lambda Ab-E and by less than 1% indirect rosetting with anti-human thymus cell antibody. A small percentage of Ig- T- 'null' cells could not be identified by either reagent. Thus, essentially pure Ig- T+ and Ig+ T- cells were readily and efficiently isolated by 'negative selection' thereby lessening the possibility of functional changes that may develop by more extensive manipulation of lymphocytes.
本文介绍了用于计数和分离Ig⁺T⁻和Ig⁻T⁺人淋巴细胞的玫瑰花结技术。为了计数Ig⁺细胞，采用了直接免疫细胞黏附技术，使用包被有纯化抗κ或抗λ轻链抗体的人红细胞（E）。κ型或λ型慢性淋巴细胞白血病显示了这些玫瑰花结的特异性。T⁺细胞通过一种新的间接玫瑰花结技术进行计数，其中淋巴细胞首先用兔抗人胸腺细胞抗血清处理，然后与包被有纯化抗兔轻链抗体的人E进行直接玫瑰花结形成。对于正常个体，使用这些玫瑰花结方法在外周血淋巴细胞以及扁桃体中发现24 - 32%的Ig⁺T⁻细胞和65 - 71%的Ig⁻T⁺细胞。Ficoll - Hypaque方法用于分别通过去除玫瑰花结化的Ig⁺细胞或T⁺细胞来获得纯化的Ig⁻T⁺和Ig⁺T⁻细胞。Ig⁻T⁺细胞的纯度通过用抗人胸腺细胞抗体（Ab）致敏的细胞大于99%的间接玫瑰花结形成以及用抗κAb - E⁺抗λAb - E的直接玫瑰花结形成小于1%来表明。所获得的Ig⁺T⁻细胞的纯度通过用抗κAb - E⁺抗λAb - E的92 - 96%的直接玫瑰花结形成以及用抗人胸腺细胞抗体的小于1%的间接玫瑰花结形成来表明。一小部分Ig⁻T⁻“裸”细胞不能被任何一种试剂识别。因此，通过“阴性选择”可以轻松高效地分离出基本上纯的Ig⁻T⁺和Ig⁺T⁻细胞，从而减少了因对淋巴细胞进行更广泛操作而可能发生功能变化的可能性。
