Expression of mJagged2 in various bone marrow fractions.

(A) Fractionation of bone marrow for RT-PCR. To purify LTR-HSC, total bone marrow was first fractionated by Nycodenz density centrifugation, followed by depletion of lineage-marker–bearing cells using monoclonal antibodies, then by FACS sorting of Rh 123-stained cells, and finally by sorting of c-kit–bearing, Ho-low cells. Additional fractions (c-kit⁻, c-kit⁺, Lin⁻c-kit⁻, Lin⁻ c-kit⁺) were sorted directly from total bone marrow or Lin⁻cells based on c-kit expression. See details in “Materials and methods.” (B) Quantitative RT-PCR for mJagged2. Serial 5-fold dilutions (up to 1:625 dilution) of cell lysates prepared from 1000 cells were used as a set in RT-PCR with nested primers. See “Materials and methods” for details of RT-PCR. Five-microliter aliquots of RT-PCR products were resolved on 2% agarose and stained with ethidium bromide. Results of β-actin RT-PCR are shown below each panel. First and last lanes of each gel contain the φχ174/HaeIII markers. (i) EML C1; (ii) unfractionated bone marrow; (iii) c-kit⁻ fraction (devoid of stem cells); (iv) c-kit⁺ fraction (containing progenitors); (v) post-Nycodenz density centrifugation (ie, MNC); (vi) Lin⁻ fraction; (vii) Lin^-− c-kit⁺ fraction (enriched for progenitors); (viii) Lin⁻ c-kit⁻fraction (devoid of stem cells); (ix) Lin⁻c-kit⁺ Rh^hi Ho^lo fraction (STR-HSC); (x) Lin⁻ c-kit⁺Rh^lo Ho^lo fraction (LTR-HSC).