Improved quantification of cell survival on stromal monolayers by flow cytometric analyses

Background: The ex vivo survival of leukemic cells maintained on bone marro w stroma is an important tool for the investigation of cell survival and le ukemogenesis. Currently, ex vivo survival of leukemic cell survival is meas ured by coculture on stromal cell monolayers. In these assays, we postulate d that two important sources of error might be introduced through either va riations in flow volume or in donor stromal cells. Methods: A previously reported coculture assay that maintains leukemic cell s on bone marrow stromal cells was employed. Results: We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties w ere added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic str omal cell assay showed a significantly lower coefficient of variation as co mpared to samples analyzed without beads or manually counted using a hemacy tometer. Second, in order to minimize variability in bone mar row hematopoi etic function between donors, an adherent stromal cell line known to suppor t hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stroma l cells derived from different donors. In contrast, statistically significa nt variability in survival of leukemic cells was not seen on HS-5 monolayer s. Finally, we demonstrate that patient-derived leukemic samples may be exa mined for cell survival using these modifications. Conclusions: The novel use of fluorescent beads and a hematopoietic-support ive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification ar e an important step in establishing a role for stromal cell assays in the s tudy of leukemia biology and therapy. Cytometry 40:26-31, 2000. (C) 2000 Wi ley-Liss, Inc.