American Society of Hematology

Fig. 2.

$Fig. 2. Isolation of TER-119+/4A5+cells from the spleens of phenylhydrazine (PHZ)-treated animals. / (A) Immunomagnetic isolation. Flow cytometric analysis of the expression of TER-119 (Y axes) and 4A5 (X axes) in nonadherent, B- and T-depleted (B−/T−), TER-119+/4A5−, and TER-119+/4A5+ cell fractions purified from the spleens of PHZ-treated mice. The gates, which identify cells expressing TER-119 and 4A5 alone or coexpressing the 2 antigens, were set to include only propidium iodide–negative cells. Gating and negative controls (not shown) are as in Figure 1A. Similar results were obtained in 7 additional purifications. The corresponding cell frequencies are summarized in Table 3. (B) Cell sorter isolation. Flow cytometric analysis of the expression of TER-119 (Y axes) and 4A5 (X axes) in light density spleen cells, in 4A5+ cells isolated during the first sorting, and in the double TER-119+/4A5+ cells isolated with the second sorting. The gates, which identify cells expressing TER-119 and 4A5 alone or coexpressing the 2 antigens, were set to include only propidium iodide–negative cells. Gating and negative controls (not shown) are as in Figure 1A. Similar results were obtained in 3 additional purifications. The corresponding cell frequencies are summarized in Table 3. (C) Analysis of immunomagnetically purified TER119+/4A5+ cells using fluorescence microscopy and image analysis. Nuclei were counterstained (blue) with DAPI. FITC-4A5 fluorescence (green) and PE-TER-119 fluorescence (red) were individually analyzed and captured (original magnification ×100). (D) Semiquantitative RT-PCR analysis of the expression of erythroid (α- and β-globin and EpoR) and megakaryocytic (AchE, Mpl, and GpIIb) genes in B−/T− cells or in TER-119+/4A5− and TER-119+/4A5+ cells (purified by double sorting) from the spleens of PHZ-treated mice. Actincomplementary DNA (cDNA) was amplified as well as control of the total cDNA used in each reaction. α-globin, GpIIb, andactin were amplified for 20, 25, and 30 cycles, whereas all the other genes were amplified for 25, 30, and 35 cycles (increasing numbers of cycles are indicated by a triangle on top of the panels). Similar results were obtained in 3 separate experiments.$

Isolation of TER-119⁺/4A5⁺cells from the spleens of phenylhydrazine (PHZ)-treated animals.

(A) Immunomagnetic isolation. Flow cytometric analysis of the expression of TER-119 (Y axes) and 4A5 (X axes) in nonadherent, B- and T-depleted (B⁻/T⁻), TER-119⁺/4A5⁻, and TER-119⁺/4A5⁺ cell fractions purified from the spleens of PHZ-treated mice. The gates, which identify cells expressing TER-119 and 4A5 alone or coexpressing the 2 antigens, were set to include only propidium iodide–negative cells. Gating and negative controls (not shown) are as in Figure 1A. Similar results were obtained in 7 additional purifications. The corresponding cell frequencies are summarized in Table 3. (B) Cell sorter isolation. Flow cytometric analysis of the expression of TER-119 (Y axes) and 4A5 (X axes) in light density spleen cells, in 4A5⁺ cells isolated during the first sorting, and in the double TER-119⁺/4A5⁺ cells isolated with the second sorting. The gates, which identify cells expressing TER-119 and 4A5 alone or coexpressing the 2 antigens, were set to include only propidium iodide–negative cells. Gating and negative controls (not shown) are as in Figure 1A. Similar results were obtained in 3 additional purifications. The corresponding cell frequencies are summarized in Table 3. (C) Analysis of immunomagnetically purified TER119⁺/4A5⁺ cells using fluorescence microscopy and image analysis. Nuclei were counterstained (blue) with DAPI. FITC-4A5 fluorescence (green) and PE-TER-119 fluorescence (red) were individually analyzed and captured (original magnification ×100). (D) Semiquantitative RT-PCR analysis of the expression of erythroid (α- and β-globin and EpoR) and megakaryocytic (AchE, Mpl, and GpIIb) genes in B⁻/T⁻ cells or in TER-119⁺/4A5⁻ and TER-119⁺/4A5⁺ cells (purified by double sorting) from the spleens of PHZ-treated mice. Actincomplementary DNA (cDNA) was amplified as well as control of the total cDNA used in each reaction. α-globin, GpIIb, andactin were amplified for 20, 25, and 30 cycles, whereas all the other genes were amplified for 25, 30, and 35 cycles (increasing numbers of cycles are indicated by a triangle on top of the panels). Similar results were obtained in 3 separate experiments.

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