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3' and 3% fetal bovine serum (FBS)] containing propidium iodide (Pl) (1 μg/ml) and analyzed on a FACS Vantage (Becton Dickinson). Residual erythrocytes, debris, doublets, and dead cells were excluded by forward scatter, side scatter, and Pl gatings as described (6).
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- cells were injected intravenously into lethally irradiated mice [9.5 gray (Gy) total body irradiation]. The spleens were removed on day 12 after injection and fixed in Bouin's solution, and macroscopically visible spleen colonies were counted.
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4 lineage-depleted BM cells were the minimum number of cells required for the survival of lethally irradiated recipients and reconstitution of their hematopoiesis. The cells in each well were collected and transferred into lethally irradiated C57BL/6-Ly5.2 mice. Several weeks to months after the transplantation, peripheral blood mononuclear cells were collected from the retro-orbital sinus and stained with FITC-anti-Ly5.1, PE-anti-myeloid (Mac-1 and Gr-1), and APC-anti-lymphoid (Thy1.2 and B220). The proportion of myeloid and lymphoid cells that originated from Ly5.1 cells was estimated by flow cytometry as described [S. Okada et al., Blood 81, 1720 (1993)].
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2, 0.2 mM dNTPs, 2.5 U of Ex Taq (Takara Ohtsu, Japan), and 50 pmol of each primer for 30 cycles (94°C, 30 s; 55°C, 30s; 72°C, 45s) followed by 5 min at 72°C in a Perkin-Elmer thermocycler. Each PCR product was electrophoresed through 2% agarose, transferred to a nylon membrane, and hybridized with a biotinylated internal oligonucleotide. Hybridized probes were detected with an ECL system (Amersham Life Sciences) according to the manufacturer's protocol.
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note
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We thank H. Kodama for discussion, T. Toyoshima for FACS operation, M. Ito for secretarial assistance, and C. Tarlinton for reading the manuscript. Supported by grants from Fujisawa Pharmaceutical, Uehara Memorial Foundation, The Ministry of Education, Science, and Culture, and the Agency for Science and Technology, Japan.
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