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Analysis of thymus derived cell sub populations by laser doppler spectroscopy and the association of electrophoretic mobility differences with differences in rosette forming affinity


, : Analysis of thymus derived cell sub populations by laser doppler spectroscopy and the association of electrophoretic mobility differences with differences in rosette forming affinity. Journal of Immunological Methods 27(3): 241-256

Rosetting procedures were used to identify and isolate human T [thymus-derived] lymphocytes having different affinities for sheep red blood cells (SRBC). High-affinity E [erythrocyte] rosette-forming cells (E-RFC) were obtained by rosetting at 29.degree. C with limited numbers of SRBC. Low-affinity and total E-RFC were obtained by rosetting at 4.degree. C with an excess of SRBC. The E-RFC isolated from these fractions on Ficoll-Hypaque gradients exhibited different electrophoretic mobilities, as measured by laser Doppler spectroscopy. The high-affinity E-RFC mobility was centered at 2.35 .mu.m/s per V per cm (for 25.degree. C, 0.28 M sucrose medium of 0.005 ionic strength), whereas the low-affinity E-RFC mobility was centered at 2.15 .mu.m/s per V per cm. The total E-RFC fractions consisted of cells of both mobilities. When Ig[immunoglobulin]G-sensitized E monolayers were used to remove cells bearing Fc receptors from mononuclear cell preparations, the non-adherent and the adherent fraction each contained a different subpopulation of T cells having mobilities centered at 2.35 and 2.15 .mu.m/s per V per cm, respectively. Rosetting assays and mobility measurements were performed simultaneously with the mononuclear cell preparations and the non-adherent agreement between the number of high-affinity E-RFC and the number of high-mobility cells. There were consistently more low-mobility cells than could be accounted for by low-affinity E-RFC. The determination of cell electrophoretic mobility was a direct physical measurement and its association with such surface markers as different affinity receptors for SRBC and Fc receptors for IgG showed that it should be a useful parameter in the characterization of lymphocytes.

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