Separation of antigen-specific lymphocytes. I. Enrichment of antigen- binding cells
Separation of antigen-specific lymphocytes. I. Enrichment of antigen- binding cells
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分离抗原特异性淋巴细胞。
DOI：
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发表时间：
1975
影响因子：
15.3
通讯作者：
J. Layton
中科院分区：
文献类型：
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作者：
W. Haas;J. Layton
将正常小鼠脾细胞分离于涂有4℃不溶的DNP-明胶或NIP-明胶薄层的培养皿中，通过37℃融化凝胶从培养皿中回收高活性细胞。NIP3-明胶层结合约0.1%的正常脾细胞，DNP4-明胶层结合约0.5%的正常脾细胞。随着吸附剂DNP浓度的增加，结合的低亲和力细胞数量增加。DNP-明胶可被DNP-赖氨酸、可溶性DNP-明胶或DNP-BSA抑制，但不能被可溶性明胶或牛血清白蛋白(BSA)抑制，因此与不溶性DNP-明胶的结合是半抗原特异性的。它也能被多价兔抗鼠Ig抑制。用125I标记的抗DNP-Ig标记DNP-明胶涂层培养皿回收的细胞表面，检测到DNP-明胶。胶原酶可以去除细胞表面结合的DNP明胶。胶原酶处理没有明显影响细胞活性或表面受体。在已知的标记B淋巴细胞的条件下，在胶原酶处理前后，90%以上的DNP-明胶结合细胞被多价125-I标记的抗鼠Ig标记。此外，经胶原酶处理后的纯化细胞群具有特异的抗原结合能力。纯化的DNP4-明胶结合细胞的DNP-RFC含量是未分离细胞的100多倍。从NIP3明胶涂层培养皿中回收的细胞群体中NIP-RFC的丰度是NIP3明胶涂层培养皿的200倍以上。
Normal mouse spleen cells were fractionated in dishes coated with thin layers of DNP-gelatin or NIP-gelatin, which were insoluble at 4 degrees C. Highly viable cells were recovered from the dishes by melting the gel at 37 degrees C. NIP3- gelatin layers bound approximately 0.1% and DNP4-gelatin layers 0.5% of normal spleen cells. Increasing numbers of low affinity cells were bound with increasing DNP density of the adsorbent. The binding to insoluble DNP-gelatin was hapten-specific since it was inhibited by DNP-lysine, soluble DNP-gelatin or DNP-BSA but not by soluble gelatin or bovine serum albumin (BSA). It was also inhibited by a polyvalent rabbit antimouse Ig. DNP-gelatin was detected on the surface of cells recovered from DNP-gelatin-coated dishes by 125- I-labeled anti-DNP Ig. The cell surface bound DNP-gelatin could be removed by treatment with collagenase. Collagenase treatment did not detectably affect cell viability or surface receptors. More than 90% of DNP-gelatin binding cells were labeled with a polyvalent 125-I-labeled antimouse Ig before or after collagenase treatment under conditions known to label B lymphocytes. Furthermore, the specific antigen-binding capacity of the purified cell populations could be demonstrated after treatment with collagenase. Purified DNP4-gelatin binding cells contained more than 100 times as many DNP-RFC than unfractionated cells. The enrichment of NIP-RFC in the cell population recovered from NIP3 gelatin-coated dishes was more than 200-fold.