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. 2013 Feb 1;8(2):e55552. doi: 10.1371/journal.pone.0055552

Figure 4. Competitive repopulation assay of HSC activity.

Figure 4

(A) Experimental scheme. Lethally irradiated B6.CD45.1 recipients were transplanted with 1000 tester LSK from WT or Id1−/− mice untreated and treated with a daily dose of 1 µg LPS for 30 days together with 1000 competitor LSK from ROSA26-stop-YFP/ROSA26-CreERT2 mice exposed to tamoxifen as described in the methods. Actions taken during the course of 168 days are indicated. (B) Analysis of primary engraftment in the bone marrow of recipients of LPS treated (black bar) and untreated (grey bar) donor LSK cells. Six weeks post transplant, mice were sacrificed and whole bone marrow (WBM) cells were collected. Donor engraftment in WBM and LSK populations were analyzed by staining with antibodies against CD45.2 donor-specific marker and determination of YFP fluorescence in CD45.2+ cells. Average percentages or numbers of donor-derived YFP negative cells are shown with SEM. (C) Analysis of secondary engraftment in the bone marrow. Pooled bone marrow from each cohort of primary recipients was transplanted into lethally irradiated recipient. Secondary recipients were sacrificed 16 weeks later. Bone marrow cells were interrogated as described for (B). (D) Analysis of peripheral blood from secondary recipients. Peripheral blood was taken 4, 12, and 16 weeks post secondary transplant. Engraftment of donor-derived YFP− cells is shown as average percentage with SEM. *** p<0.001; *p<0.05.