![Fig. 2. Isolation and functional characterization of Rhlo and Rhhi cell populations. / (A) A cartoon showing 2 important populations of cells: LT-HSCs and multipotent progenitors (MPPs). The LT-HSCs can support long-term (up to 6 months) hematopoiesis in competitive repopulation assay. MPP cells can only briefly support (< 4 weeks) hematopoiesis in the same assay. Rhodamine-123 (Rh) was used to separate these 2 populations of cells. (B) Separation of RhloLin−/loSca-1+c-kit+and RhhiLin−/loSca-1+c-kit+cells using flow cytometry. R3: Lin−/loSca-1+c-kit+ cells; R2: Rhlo (15% of cells with the lowest staining); R4: Rhhi (15% of cells with the highest staining). (C) Competitive repopulation assay. Either RhloLin-/loSca-1+c-kit+or RhhiLin-/loSca-1+c-kit+cells (250 cells) derived from the donor Ly5.2 mice were mixed with 4 × 104 Lin−/lo cells from congenic host strain Ly5.1,17 and injected to lethally irradiated (11 Gy [1100 rads]) Ly5.1 mice. Eight mice were used per group. Peripheral blood cells were harvested at different time points after transplantation as indicated. Flow cytometric analyses were performed using different specific cell surface markers (CD3, B220, Mac-1, and Gr-1) together with anti-Ly5.2 antibody. ○ indicates Rhlo lymphoid lineage; ▵, Rhlo myeloid lineage; ●, Rhhi lymphoid lineage; ▴, Rhhi myeloid lineage.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/2/10.1182_blood.v99.2.488/6/h80222024002.jpeg?Expires=1735569416&Signature=mPAfc1vvQcC9dqqMb0nNQ24xbjkeqrk-fl6sNiKNJYN42YoOl36rdDtm1W7bClwhYDZkFrMDC3T7xX8JLZdZvVQMSH8NwQ4D~j1r3-16zpu-v8MOA~O-SjgblZKnm0RjdoEVb3JnUq21PaUVudjNvu~GNuMWNUefYhs26TuWSkviE76EoTkWuyv88A4fpeS7enyP60KmlA1AERR25rNmrQ2~TQG3PaQEy0YJLyeSz~47PPLTXrG7JhK7aspvAHuLc0p6jGk0iEG16qe6yCFFViibuF1CB0HBn-SFXkz~QPzQT3SxlJcec9HzA46YdPh7EPApR91~RmYrofzRhNd8oA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Isolation and functional characterization of Rhlo and Rhhi cell populations.
(A) A cartoon showing 2 important populations of cells: LT-HSCs and multipotent progenitors (MPPs). The LT-HSCs can support long-term (up to 6 months) hematopoiesis in competitive repopulation assay. MPP cells can only briefly support (< 4 weeks) hematopoiesis in the same assay. Rhodamine-123 (Rh) was used to separate these 2 populations of cells. (B) Separation of RhloLin−/loSca-1+c-kit+and RhhiLin−/loSca-1+c-kit+cells using flow cytometry. R3: Lin−/loSca-1+c-kit+ cells; R2: Rhlo (15% of cells with the lowest staining); R4: Rhhi (15% of cells with the highest staining). (C) Competitive repopulation assay. Either RhloLin-/loSca-1+c-kit+or RhhiLin-/loSca-1+c-kit+cells (250 cells) derived from the donor Ly5.2 mice were mixed with 4 × 104 Lin−/lo cells from congenic host strain Ly5.1,17 and injected to lethally irradiated (11 Gy [1100 rads]) Ly5.1 mice. Eight mice were used per group. Peripheral blood cells were harvested at different time points after transplantation as indicated. Flow cytometric analyses were performed using different specific cell surface markers (CD3, B220, Mac-1, and Gr-1) together with anti-Ly5.2 antibody. ○ indicates Rhlo lymphoid lineage; ▵, Rhlo myeloid lineage; ●, Rhhi lymphoid lineage; ▴, Rhhi myeloid lineage.
