Flow Cytometric Analysis

3.7.1. First Step Screening

As a first step, all wells were screened for the generation of cells from the seeded single Ly5.1 progenitor. For the first screening, cells were stained with two colors with anti-Ly5.1 and anti-Ly5.2. The samples containing Ly5.1+ cells are selected for further analysis.

3.7.2. Staining of Cells for Second-Step Analysis

Samples selected for further analysis are stained with four colors using the combination of anti-Ly5.1, anti-Thy-1, anti-B220, and anti-Mac-1. Staining with three colors using a combination of: 1) anti-Ly5.1, anti-Thy-1, anti-B220, and 2) anti-Ly5.1, anti-Thy-1, and anti-Mac-1 also works.

3.7.3. Judgment of Progenitor Types

Any lineage markers would give some non-specific staining, so the judgment of lineage type of generated cells requires evaluation from several angles. A large gate area is set in forward/side scatter in order to include all viable cells (Fig. 6A). Cells showing Ly5.1+Thy-1+B220- and Ly5.1+Thy-1B220+phenotypes are tentatively regarded as donor derived T- and B-cells, respectively. Cells in these populations are then checked for forward/side scatter and expression of Mac-1, and those falling within the lymphocyte area of scattergram and negative for Mac-1 expression are judged to be T or B lineage cells. Ly5.1+Mac-1+ cells with myeloid scatter characteristics are considered to be donor derived myeloid lineage cells. Note that these Mac-1+ cells sometimes include B220 low positive or Thy-1 low positive cells. The progenitor type is determined based on the types of cells generated. The progenitor generating T, B and myeloid lineage cells is termed p-Multi. Likewise, other types are termed p-MT, p-MB, p-TB, p-M, p-T, and p-B (see Note 11). Among these types, p-TB has never been detected in fetal tissues (see Note 12). Representative flow cytometric profiles of cells derived from each of the six types of progenitors are shown in Fig. 6B.

3.7.4. The Distribution of Different Types of Progenitors in Fetal Liver Subpopulations

An example of the distribution of different progenitor types found in fetal liver is shown in Fig. 7. As a progenitor source, Sca-1+ and Sca-1- cells from the Linc-kit+ population of 12 dpc murine fetal liver were used (Fig. 7A). 124 cells of the Sca-1+ group and 160 cells of the Sca-1- group were examined (see Fig. 7B; ref. 19; Note 13).

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