Research Article

Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms

Figure 6

Optimized protocol for isolation of rVSELs from WT and eGFP-expressing rat BM tissue. Rat BM-derived VSELs were isolated from full population of BM cells stained for CD45 (PE-Cy7), Lin markers (TCRαβ, CD3, CD11b, and CD45RA; Alexa Fluor 647), and CD106 (PE) by MoFlo XDP cell sorter (Beckman Coulter). (a) Total nucleated cells (TNCs) are visualized on dot-plot showing FSC-H versus SSC-H. (b) Small agranular cells from gate R1 (with extension of lymphgate into low values of FSC; Figure 5(a)) are plotted on FSC-W versus FSC-H dot-plot to exclude doublets. (c) Single cells from gate R2 are subsequently analyzed for Lin markers expression. (d) Lin events included in region R3 are further plotted on dot-plot showing CD106 expression versus side scattered characteristics (SSC-H) of these cells. (e) CD106+ cells from gate R4 are eventually visualized on dot-plot based on their CD45 expression and FSC signal (FSC-H). Rat HSCs are identified as CD45+/Lin/CD106+ (region R6), while rVSELs as //CD45/Lin/CD106+ cells (region R5).