Fig. 5. Dynamic analysis of Lin^- bone marrow cultured for 3 days under improved erythropoieitic conditions: flow cytometry and benzidine-Giemsa staining. Purified Lin^- cells were cultured in vitro for 3 days on fibronectin-coated plates in medium containing serum. Epo, SCF, Dex, and IGF-I included in the medium for the first day of culture, and then the medium was changed to remove soluble growth factors. The differentiation profile of the cultured cells was examined by both flow cytometry and benzidine-Giemsa stain after each day in culture. Two representative micrographs of these stained populations are presented from each day of erythropoieitc culture. After the third day, flow cytometry indicated that much of the cultured population expressed Ter-119 and CD71. Furthermore, benizidine-Giemsa stain revealed that many cells in the harvested population were enucleated and expressing hemoglobin. The arrowhead indicates a hemoglobin⁺ normoblast, and the arrow indicates an enucleated reticulocyte. (Scale bars, 20 mm.)

Fig. 6. Growth and cell-cycle dynamics during improved erythropoietic culture. Erythropoietic cultures were initiated with WT Lin^- BM and then harvested at various times to assess growth and cell cycle status. (A) Viable cell numbers were measured by using a ViCellXR viable cell counter, and were then normalized to input cell numbers. The log₂ of this ratio was then taken to estimate the total number of doublings that had occurred at a given culture time. The data are presented as the mean of four biologically independent samples, obtained over the course of two experiments, ±SD. Whereas growth on days 1 and 3 resulted in a single doubling of cell numbers, growth on day 2 resulted in more than two doublings. At harvest, »30% of viable cells are PCEs, and viable cell numbers have increased by a factor of ~2^4.2. Given that »5 ´ 10⁵ Lin^- BM cells are obtained per mouse, and that 2,000 PCEs are sufficient to detect genotoxicity by in vitro erythroid MN formation, it is estimated that >1,000 in vitro erythroid MN assays can be conducted by using the hind-leg BM of a single mouse. (B) Biologically independent samples were harvested at various points in erythropoietic culture (please see the table for n). The harvested populations were washed, fixed in cold ethanol for >1 h, washed again, and then stained for cell cycle analysis using propidium iodide (50 mg/ml) in the presence of RNaseA (500 mg/ml). DNA content was measured for each cell by using flow cytometry, and then the ModFit LT diploid model was used to fit the resulting flow cytometry histograms. These results from cell cycle analysis are tabulated here for various times in erythropoietic culture. Consistent with the rapid increase in viable cell numbers during the second day of cell culture (see A), most cells were found to be in S phase during this period.

Fig. 7. In vitro erythropoiesis continues after treatment with alkylating agents: flow cytometry and benzidine-Giemsa cytology. The cultured populations analyzed for Fig. 2 were also examined by flow cytometry and benzidine-Giemsa staining. Two representative micrographs of populations treated with each model genotoxciant are presented. Cytology revealed that large fractions of the treated populations were hemoglobin⁺ and fully enucleated. In addition, large fractions of the treated populations were found by flow cytometry to express the characteristic late-erythroid surface markers Ter-119 and CD71. The arrowheads indicate hemoglobin⁺ cells containing micronuclei. (Scale bars, 20 mm.)

Fig. 8. Dynamic analysis of MGMT^-/- Lin^- bone marrow cultured for 3 days under improved erythropoieitic conditions: flow cytometry and benzidine-Giemsa staining. Purified Lin^- cells from MGMT^-/- mice were cultured in vitro for 3 days on fibronectin-coated plates in medium containing serum. Epo, SCF, Dex, and IGF-I were included in the medium for the first day of culture, and then the medium was changed to remove soluble growth factors. The differentiation profile of the cultured cells was examined by both flow cytometry and benzidine-Giemsa stain after each day in culture. Two representative micrographs of these stained populations are presented from each day of erythropoietic culture. After the third day, flow cytometry indicated that much of the cultured population had acquired a late erythroid surface phenotype during culture. Furthermore, benizidine-Giemsa stain revealed that many cells in the harvested population were enucleated and expressing hemoglobin. The arrowhead indicates a hemoglobin⁺ normoblast, and the arrow indicates an enucleated reticulocyte. (Scale bars, 20 mm.)

Fig. 9. Early BCNU treatment of WT Lin^- BM cultures leads to decreased viable cell numbers and diminished MN formation. Erythropoietic cultures initiated with WT Lin^- BM were treated with BCNU 4 h after culture was initiated. Seventy-two hours later, the cultures were harvested, and genotoxic effects were quantified both by: (A) cytological scoring of MN frequency and (B) viable cell counts. The methods used to generate these data were identical to those used to generate Fig. 4, except that treatment of BCNU was delivered 4 h after cultures were initiated. By comparison with Fig. 4, these data further support the noted trend that treatment earlier in the erythropoietic culture period leads to a lower frequency of micronucleated PCEs and a lower number of viable cells upon harvest. *, Significant difference from untreated control (P < 0.05).