Relative enrichment of hematopoietic progenitor cells: efficiency of a repeated density centrifugation.
Low-density cells were prepared from 11 bone marrow samples by centrifugation on Ficoll-sodium diatrizoate. Repeated density gradient centrifugation of the cells collected from the interface of the first gradient removed most nonnucleated erythroid cells. A mean of 47.9% (19.4% to 76.0%) of the mononuclear cells collected after the initial centrifugation were recovered from the interface of the second gradient, whereas 13.3% (3.7% to 34.9%) of the MNCs were counted in the high-density pellet and 38.9% (3.8% to 65.7%) of the MNCs were lost unspecifically. In contrast, a mean of 71.7% (43.0% to 91.3%) of the colony-forming units were recovered from the interface after the second centrifugation (as determined by colony formation assays), whereas only 3.2% (0.5% to 7.0%) were found in the high-density pellet. The unspecific loss of colony-forming units was 25.1% (1.7% to 51.4%). The results demonstrate a relative enrichment of colony-forming units in the culture assay by 1.7 times (average). The method is recommended as an additional preparative step before fluorescence-activated sorting of viable cells, because removal of most erythrocytes and late normoblasts strongly reduces the time required for sorting.
Children's Cancer Research Institute, St. Anna Children's Hospital, Vienna, Austria.
G
G
Fritsch
P
P
Buchinger
D
D
Printz
H
H
Gadner
D000293:Adolescent; D000328:Adult; D001854:Bone Marrow Cells; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000592:standards*; D002648:Child; D002675:Child, Preschool; D005260:Female; D005434:Flow Cytometry; D005453:Fluorescence; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007223:Infant; D008297:Male