Fig. 3.
Fig. 3. Four-color flow cytometry analysis of T and B lymphocyte subpopulations in the thymus, BM, and lymph node of chimeric mice. The mice were killed at 2 or 8 months after injection of HSCs. Gate used to define donor-derived cells were set according to the expression of Ly5.2 and Ly5.1. Results shown are representative of eight experiments. B cell subpopulations in BM (A) and lymph node (B) of chimeric mice. Single cell suspensions of BM cells were stained with donor-specific MoAb (anti-Ly5.2), B220, Gr-1, and IgM. The two-color contour plots of the expression of B220 and Gr-1 are shown (A, upper) and expression of B220 and IgM is shown (A, lower). Two color contour plots of B220 and IgM expression analyzed at 2 months (upper) and 8 months (lower) are shown in B. CD4 and CD8 expression on donor-derived cells in thymus (C) or lymph node (D). Two-color contour plots from analysis at 2 months after injection (upper) and 8 months after injection (lower) are shown. / (E) Short-term T-cell development in the thymus of recipient animals. Purified HSCs from bcl-2−/− or +/− BM are injected into 6.5 Gy irradiated RAG-2 KO recipients, and analyzed for CD4, CD8, or TCRαβ expression on donor-derived cells in thymus at indicated periods after transplantation. Two-color contour plots of CD4 and CD8 expression (upper) or single color histogram of TCRαβ expression (lower) are shown. (F ) PCR analysis of D-Jβ and V-DJβ gene rearrangements in the thymocytes. Genomic DNA prepared from thymocytes of indicated recipient animals was subject to the gene rearrangement analysis using indicated PCR primers and probes, as describe in Materials and Methods. In all experiments measuring DNA rearrangements, equal amounts (0.5 μg/reaction) of genomic DNA were used for PCR amplification to minimize possible deviation among multiple PCR reactions. The sensitivity for every PCR detection was normalized by pre-titrating PCR cycle numbers. For the analysis of D-Jβ rearrangement, unrearranged DNA gives 1.8 kb signals corresponding to germline D-Jβ2, whereas the rearrangement gives six discrete signals corresponding to rearranged D-J genes using six Jβ2 segments. For the analysis of Vβ8.2-DJβ, unrearranged DNA gives no signals, whereas the rearrangement gives discrete signals corresponding to rearranged genes. Biotinylated Hinf I fragments of pX174 DNA (726-24 bp; GIBCO-BRL) were used as a molecular weight marker.

Four-color flow cytometry analysis of T and B lymphocyte subpopulations in the thymus, BM, and lymph node of chimeric mice. The mice were killed at 2 or 8 months after injection of HSCs. Gate used to define donor-derived cells were set according to the expression of Ly5.2 and Ly5.1. Results shown are representative of eight experiments. B cell subpopulations in BM (A) and lymph node (B) of chimeric mice. Single cell suspensions of BM cells were stained with donor-specific MoAb (anti-Ly5.2), B220, Gr-1, and IgM. The two-color contour plots of the expression of B220 and Gr-1 are shown (A, upper) and expression of B220 and IgM is shown (A, lower). Two color contour plots of B220 and IgM expression analyzed at 2 months (upper) and 8 months (lower) are shown in B. CD4 and CD8 expression on donor-derived cells in thymus (C) or lymph node (D). Two-color contour plots from analysis at 2 months after injection (upper) and 8 months after injection (lower) are shown.

(E) Short-term T-cell development in the thymus of recipient animals. Purified HSCs from bcl-2−/− or +/− BM are injected into 6.5 Gy irradiated RAG-2 KO recipients, and analyzed for CD4, CD8, or TCRαβ expression on donor-derived cells in thymus at indicated periods after transplantation. Two-color contour plots of CD4 and CD8 expression (upper) or single color histogram of TCRαβ expression (lower) are shown. (F ) PCR analysis of D-Jβ and V-DJβ gene rearrangements in the thymocytes. Genomic DNA prepared from thymocytes of indicated recipient animals was subject to the gene rearrangement analysis using indicated PCR primers and probes, as describe in Materials and Methods. In all experiments measuring DNA rearrangements, equal amounts (0.5 μg/reaction) of genomic DNA were used for PCR amplification to minimize possible deviation among multiple PCR reactions. The sensitivity for every PCR detection was normalized by pre-titrating PCR cycle numbers. For the analysis of D-Jβ rearrangement, unrearranged DNA gives 1.8 kb signals corresponding to germline D-Jβ2, whereas the rearrangement gives six discrete signals corresponding to rearranged D-J genes using six Jβ2 segments. For the analysis of Vβ8.2-DJβ, unrearranged DNA gives no signals, whereas the rearrangement gives discrete signals corresponding to rearranged genes. Biotinylated Hinf I fragments of pX174 DNA (726-24 bp; GIBCO-BRL) were used as a molecular weight marker.

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