Journal of Experimental Medicine, Vol 141, 1004-1014, Copyright © 1975 by Rockefeller University Press

ARTICLES

Separation of antigen-specific lymphocytes. I. Enrichment of antigen- binding cells

W Haas and JE Layton

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.
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This article has been cited by other articles:

• Scott, D.W., Ales-Martinez, J.-E., Chace, J.H., LoCascio, N.J., Silver, L., Warner, G.L. (1989). Models of B-cell Unresponsiveness. Cold Spring Harb Symp Quant Biol 54: 899-905 [Abstract]  
• Nossal, G. J. V., Pike, B. L., Stocker, J. W., Layton, J. E., Goding, J. W. (1977). Hapten-specific B Lymphocytes: Enrichment, Cloning, Receptor Analysis, and Tolerance Induction. Cold Spring Harb Symp Quant Biol 41: 237-243 [Abstract]  

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