Serial 5FU treatment and competitive BM reconstitution assay

Eight-week-old female B6.SJL-Ptprc^a/BoyAiTac (CD45.1) mice were treated with 4 rounds of vehicle (1x phosphate buffered saline [PBS]) or 150 mg/kg 5FU at 3-week intervals. A subset of the 5FU-treated mice was given a single dose of 150 mg/kg G1T28 by oral gavage 30 minutes before each 5FU injection or 2 doses of 250 μg/kg pegfilgrastim by SC injection at 24 and 48 hours after each 5FU treatment, and the remaining mice received vehicle (50 mM citrate buffer (pH4.0) for gavaging and 0.1% bovine serum albumin in 1x PBS for SC injection) treatment. CBCs were analyzed 4, 7, 11, 14, 18, and 21 days after the last round of 5FU treatment to assess the rate of hematologic recovery. Eight weeks after the last dose of 5FU treatment, BM cells were harvested from the treated mice, and competitive long-term BM reconstitution assay was performed by transplanting 500,000 total BM cells (CD45.1⁺) from each donor, together with 500,000 competitor BM cells (CD45.2⁺, from 8-week-old C57BL/6 mice), into lethally irradiated 8-week-old female C57BL/6 (CD45.2⁺) recipients. Lethal irradiation was carried out using a Gammacell 40 Exactor Cesium137 γ-ray source (MDS Nordia) by giving two doses of 540 rads total body irradiation 2 hours apart. BM cells from each donor mouse were transplanted into 5 recipient mice, and 5 donors were transplanted from each treatment group. PB was collected from the tail veins of recipient mice at 4-week intervals for at least 16 weeks after transplantation. To assess the frequency of donor cells in PB, red blood cells were subjected to ammonium-chloride potassium lysis (54), and remaining leukocytes were stained with antibodies against CD45.2 (104, FITC), CD45.1 (A20, APC-Efluor 780, eBioscience), B220 (PerCP-Cy5.5), Mac-1 (APC), CD3e (PE), and Gr-1 (PE-Cy7). Thirty two weeks after initial transplantation, BM cells were harvested from each primary recipient mouse and analyzed for donor cell contribution to each hematopoietic lineage. For HSC analysis, BM cells were stained with antibodies against lineage markers (FITC), c-Kit (APC-Efluor 780), Sca-1 (PE-Cy7), CD150 (PerCP-Cy5.5), CD48 (PE), CD45.1 (BV421), and CD45.2 (Biotin, followed by streptavidin-Alexa Fluor 647). For analysis of mature myeloid, B, and T cells, BM cells were stained with antibodies against Mac-1 (APC-Efluor 780), Gr-1 (PE-Cy7), B220 (PerCP-Cy5.5), CD3 (PE), CD45.2 (FITC), and CD45.1 (BV421). Secondary BM transplantation was performed by transplanting 1 × 10⁷ total BM cells from each primary recipient into an individual lethally irradiated secondary recipient (CD45.2⁺). The frequencies of donor CD45.1⁺ cells in each blood lineage were monitored by analyzing the PB of secondary recipients at 4-week intervals for at least 16 weeks after transplantation.