Identification of hybridomas derived from mouse CD5+ B lymphocytes by fluorescent staining for cytoplasmic CD5 expression.
Identification of hybridomas derived from mouse CD5+ B lymphocytes by fluorescent staining for cytoplasmic CD5 expression.
复制标题
通过细胞质 CD5 表达荧光染色鉴定源自小鼠 CD5 B 淋巴细胞的杂交瘤。
作者：
S. Sadigh;B. B. Scott;R. Mageed;A. Malcolm;E. Andrew;R. Maini
CD5+B淋巴细胞杂交瘤的产生对研究CD5+B淋巴细胞亚群的免疫球蛋白基因谱和抗体特异性具有重要意义。然而，来源于这些淋巴细胞的杂交瘤在杂交后总是失去CD5的表面膜表达。这阻碍了产生的杂交瘤从未分选或部分分选的细胞中明确地分配给CD5+B淋巴细胞亚群。最近的研究表明，在一些小鼠杂交瘤的细胞质中可以检测到CD5基因的mRNA转录本和蛋白产物，这表明虽然表面表达已经丢失，但它们可能是从CD5+B淋巴细胞产生的。然而，这些研究无法完全排除CD5基因胞质异常表达是杂交过程的直接结果的可能性。为了证实杂交瘤细胞胞浆CD5的表达实际上与B淋巴细胞的来源直接相关，我们从FACS分选的CD5+和CD5-小鼠脾B淋巴细胞中制备了杂交瘤细胞，并用荧光激活细胞分选法测定了它们的表面和胞浆CD5的表达。结果表明，CD5+B淋巴细胞杂交瘤(9株)表面CD5表达均为阴性，胞浆CD5表达均为阳性，而CD5-B淋巴细胞杂交瘤(9株杂交瘤)表面和胞浆CD5表达均为阴性。这一发现表明，胞浆CD5表达的染色为确定小鼠B淋巴细胞杂交瘤的细胞来源提供了一种准确的方法。
The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immunoglobulin gene repertoire and antibody specificity of this B-lymphocyte subpopulation. However, hybridomas derived from these lymphocytes invariably lose surface membrane expression of CD5 following hybridization. This impedes the unequivocal assignment of the generated hybridomas to the CD5+ B-lymphocyte subpopulation from unsorted, or partially sorted, cells. Recent studies have shown that mRNA transcripts of the CD5 gene and the protein product can be detected in the cytoplasm of some mouse hybridomas, indicating that although surface expression has been lost, they may have been generated from CD5+ B lymphocytes. These studies, however, have been unable to discount completely the possibility that aberrant cytoplasmic expression of the CD5 gene occurred as a direct consequence of the hybridization process. To confirm that cytoplasmic CD5 expression in the hybridomas is in fact directly related to the B-lymphocyte origin we have generated hybridomas from FACS-sorted CD5+ and CD5- murine splenic B lymphocytes, and determined their surface and cytoplasmic CD5 expression by fluorescence-activated cell sorting. Our findings reveal that all hybridomas derived from CD5+ B lymphocytes (nine hybridomas) were negative for surface but positive for cytoplasmic CD5 expression, whereas all hybridomas derived from CD5- B lymphocytes (nine hybridomas) were negative for both surface and cytoplasmic CD5 expression. This finding shows that staining for cytoplasmic CD5 expression provides an accurate method of determining the cellular origin of murine B-lymphocyte hybridomas.