(A) LSKs were fractionated according to their Flt3 and CCR9-EGFP expression

The FACS plot on the left is gated on Lin cells, and the plot on the right is gated on LSK cells (as shown in the plot on the left). LSK subpopulations were sorted from the indicated regions. Percentages are indicated. (B) 100 cells of the indicated precursors were sorted onto OP9-DL1 stroma, cultured for 9 d, and analyzed by FACS. The levels of CCR9-EGFP and CD44 expression are indicative of the maturity of each precursor, as T lineage–biased precursors are expected to progress faster, whereas precursors without a T lineage bias or those biased toward a non-T fate will take longer to develop to DN3 thymocytes. The histogram plots are gated on DN2/3 thymocytes in which CCR9-EGFP expression increases with the progression from DN2 to DN3 thymocytes (reference ). The results of three independent wells are shown in each histogram plot. Contour plots are gated on Lin CD90 cells, and the numbers in each quadrant indicate the mean frequency of each population from three independent experiments. (C) The results of a representative clonogenic myeloid progenitor assay are shown for a 96-well plate seeded with 5, 10, 20, or 40 cells of the indicated precursors. The number of detectable myeloid colonies for each well is given. (D) Semiquantitative RT-PCR was performed on fivefold dilutions of cDNA prepared from the indicated precursors with primers specific for the indicated transcripts. Primers specific for hypoxanthin-phosphoribosyl-transferase (HPRT) transcript were used as a loading control. PCR fragment sizes in base pairs are given in parenthesis.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

The LSK population was analyzed by FACS in CCR9/ //Flt3∷Cre//R26R-EYFP mice

The top left plot is gated on LSK bone marrow cells, and the histogram plots are gated on the indicated regions of interest. The fraction of EYFP, recombined cells is shown for each subset. Percentages are indicated.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

(A) Lin blood cells of heterozygous CCR9-EGFP knock-in mice were fractionated according to their CD117 and CCR9 expression

The plot is gated on Lin cells. Percentages are indicated. (B) Lin fractions with the indicated phenotypes were isolated from blood (as shown in A) and from bone marrow, and were cultured as groups of 100 cells on OP9-DL1 stromal layers for 12 and 19 d. The fraction of wells that contained T lineage–committed DN3/4 thymocytes is plotted for each time point. At least 1,200 cells cultured as 100 cells per well were assayed for each population and time point. For the analysis of T lineage precursor activity of Lin CD117 CCR9 blood cells at day 12, >2,000 cells cultured as 100 cells per well were analyzed.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

(A) In the absence of Notch1, only very few ETPs can be isolated for the detection of T cell lineage precursors

The FACS plots are gated on Lin CD44 CD25 DN1 thymocytes derived from Notch1//CCR9/+ and Notch1//MxCre//CCR9/+ mice 2 wk after pIpC injection. Percentages are indicated. (B) Single TMPs were sorted from Notch1//CCR9/+ and Notch1//MxCre//CCR9/+ mice 4 wk after pIpC injection on OP9-DL1 stromal layers and cultured for 12–14 d. Cells falling into the right half of the ETP gate shown in A were considered TMPs. DN3/4 thymocytes developed on OP9-DL1 stroma even in the absence of Notch1, as previously described (reference ). Percentages are indicated. (C) Genomic PCR was performed on DNA purified from the OP9-DL1 culture of DN3/4 thymocytes generated from a single TMP of a Notch1//CCR9/+ and a Notch1//MxCre//CCR9/+ mouse 4 wk after pIpC injection. Two sets of primers were used, with one detecting the wild-type (N1 wild-type) and the floxed (N1 allele) Notch1 allele (left lane) and the other detecting the deleted allele of Notch1 (Δ N1; right lane). Because the added hematopoietic cells did not carry a wild-type Notch1 allele, this band is thought to be derived from OP9-DL1 stroma. PCR fragment sizes in base pairs are given in parenthesis.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p