High throughput cell-based assay of hematopoietic progenitor differentiation.
The in vitro efficacy of drug candidates relative to hematopoietic stem cell proliferation and differentiation is currently assayed through use of the clonogenic "colony assay." The extremely low throughput of this assay precludes its use in library screening and much drug discovery work. A rapid-throughput assay of progenitor cell differentiation based on the quantification of hematopoietic lineage-specific markers has been developed. The CELISA assay employs a single incubation with a lanthanide-conjugated primary antibody and subsequent time-resolved fluorescence spectroscopy. The rapid-throughput nature of this assay is enhanced by the use of cell culture-compatible filter plates to reduce the number of manipulations as compared to currently available cell-based assays. The culture and assay are done in 96-well plates, and the quantitation process requires approximately 1 hour. The myeloid, erythroid, and megakaryocytic lineages can be objectively quantified; data from the assay correlate extremely well with data generated through use of the traditional colony assay. This assay makes possible both rapid-throughput drug discovery and toxicity screening in the area of hematopoiesis.
Greenwalt
D E
DE
BioWhittaker, Inc., Walkersville, MD 21793-0127, USA. dale.greenwalt@biowhittaker.com
Szabo
J
J
Manchel
I
I
D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology; D002454:Cell Differentiation; D002478:Cells, Cultured; D015925:Cryopreservation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans
D018952:Antigens, CD34