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Abstract
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.