Fractionation of normal and leukemic T-cells by lectin-affinity column chromatography.
A method for rapid fractionation of normal and leukemic T-cells (Jurkat, RPMI-8402, MOLT-4), using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently conjugated with Dolichos biflorus agglutinin (DBA) over 24 h. The normal cells were eluted by phosphate buffered saline (Ca(2+) and Mg(2+) free), while the leukemic T-cells, interacting with DBA, were removed by N-acetyl-D-galactosamine or by low-concentrated acetic acid as a mobile phase. The cell fractions were detected spectrophotometrically at 600 nm. The rate of cell elution decreased in the order: normal>leukemic T-cells. The viability and the type of separated T-cell fractions were characterized by flow cytometry, using adequate fluorescent antibodies. The interactions between leukemic T-cells and DBA-saturated Sepharose beads were examined by fluorescent microscopy, using fluorescent isothiocyanate-DBA as a fluorescent marker.
Natural Substance-Composed Materials Group, Institute for Structural and Engineering Materials, Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, AIST-Kyushu, 807-1 Shuku, Tosu, Saga-ken, Japan.
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Ohba
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Bakalova
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Moriwaki
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Nakamura
D015703:Antigens, CD / Q000032:analysis; D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis; D019169:Jurkat Cells; D037102:Lectins; D008856:Microscopy, Fluorescence; D012016:Reference Values; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000473:pathology; D018800:Thy-1 Antigens / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000276:immunology / Q000473:pathology*
D015703:Antigens, CD; D018960:Hyaluronan Receptors; D037102:Lectins; D018800:Thy-1 Antigens