Magnetic activated cell sorting (MACS)--a new immunomagnetic method for megakaryocytic cell isolation: comparison of different separation techniques.
Megakaryocytes are difficult to isolate because of their fragility, their tendency to aggregate, and their varying sizes. For purification of cells at different stages of maturation and of different sizes (ploidy classes) we developed an immunomagnetic cell sorting method (MACS) to enrich the whole spectrum of the megakaryocytic cell lineage. The use of small magnetic beads coupled to various antibodies and labelling with fluorescent antibodies allowed direct analysis of enrichment and evaluation of the isolated fraction without further staining or detachment procedures. CD 61 (Y2/51), a monoclonal antibody directed against platelet glycoprotein IIIa, was employed to perform the separation procedure. An enrichment up to 47% of CD 61-positive cells with an average of 37% and a recovery rate of 37% was obtained by using the MACS technique. Pre-enrichment by Percoll density centrifugation, followed by MACS separation, resulted in an enrichment of 65% and a recovery rate of 67%. The relative amount of small megakaryocytic cells in only MACS-enriched cell populations, however, was higher than in Percoll/MACS fractions. As a parameter of vitality we tested cytokine secretion of the enriched megakaryocytes in reverse haemolytic plaque assays. Secretion of IL-1, IL-6, GM-CSF, and PDGF with and without stimulation by phorbol myristate acetate was demonstrable at the single cell level.
Institute of Pathology, University of Cologne, Germany.
B
B
Schmitz
A
A
Radbruch
T
T
Kümmel
C
C
Wickenhauser
H
H
Korb
M L
ML
Hansmann
J
J
Thiele
R
R
Fischer
D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D016207:Cytokines / Q000187:drug effects / Q000378:metabolism; D006462:Hemolytic Plaque Technique; D006801:Humans; D018189:Immunomagnetic Separation; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016207:Cytokines; D013755:Tetradecanoylphorbol Acetate