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5 cells per well were transferred onto fresh OP9 cells. Total cells of individual wells were harvested by vigorous pipetting and transferred to a fresh OP9 cell layer on individual wells again at day 10. Erythroid lineage cells were counted by 2,7-diaminofluorene or May-Giemsa staining or both, and the two counts were in general agreement (11).
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Cytospin specimens of day 7 or day 14 cells were stained with May-Giemsa solution The specimens were fixed with cold methanol and stained by peroxidase-antiperoxidase (PAP) immunoenzymatic staining kit as recommended by the manufacturer (DAKO, Carpinteria, CA). Rabbit anti-mouse embryonic hemoglobin (1/300 dilution) (11) and rabbit anti-mouse adult hemoglobin (Cappel Research Products, Durham, NC) were used as the primary antibodies.
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Virtually all of the erythroid lineage cells on days 6 and 7 were positive for staining with antisera to embryonic hemoglobin. In contrast, less than 1% of erythroid lineage cells contained embryonic hemoglobin after day 10 in the absence of EPO The relative frequency of EryP and EryD at days 8 and 9 could not be determined accurately because of small numbers of total erythroid lineage cells.
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Day 4-induced ES cells were trypsinized and transferred onto an OP9 cell layer Three days after the transfer, individual, well-separated, adherent small round cell clusters were picked and put en bloc into methylcellulose media containing EPO and interleukin-3 (18). Five to 7 days later, emerged colonies were picked, washed, and divided into three portions. Cytospin specimens were produced from the three portions. One portion was stained with May-Grünwald Giemsa, and the two other portions were stained with the PAP method by use of rabbit anti-mouse embryonic hemoglobin or rabbit anti-mouse adult hemoglobin as the primary antibodies (10)
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13344273399
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5 day 5-induced cells Standard errors are not indicated but are located within each circle. In some experiments, Ack2 (10 μg/ml) or human recombinant EPO (10 U/ml) were added
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13344255931
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note
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Culture plates that were seeded with day 4-induced cells were examined 4 days after the transfer as described in Table 2 However, appearance of EryP in culture plates seeded with day 3-induced cells was examined microscopically 3 days after the transfer without dianisidine staining. This microscopic assay was relatively easy to perform because more than 90% of nonadherent cells were EryP (15) Day 3 cell cultures were continued for another 2 days to examine subsequent appearance of adherent hematopoietic cell clusters microscopically. This step was necessary because a large number of EryP appeared that originated from secondarily differentiated nonhematopoietic colonies when we waited until 5 days after the transfer (15).
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13344273400
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note
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We thank S Okazaki for technical assistance, S-i. Nishikawa for Ack2 antibody to c-Kit, T. Atsumi to rabbit anti-mouse embryonic hemoglobin, and Kinn Brewery for human recombinant EPO reagents. Supported by grants from Vehara Memorial Foundation, Kanae Foundation of Research for New Medicine, and the Ministry of Education, Science, Sports and Culture of Japan.
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