Bertoncello Ivan, Williams Brenda
Stem Cell Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
Methods Mol Biol. 2004;263:181-200. doi: 10.1385/1-59259-773-4:181.
A dual-dye efflux strategy utilizing the supravital dyes Hoechst 33342 (Ho) and rhodamine 123 (Rh123) is described and illustrated for the detection and analysis of hematopoietic stem cells in murine bone marrow. Mononuclear cells from bone marrow cell suspensions were incubated in a cocktail of Rh123 plus Ho, and both dyes were effluxed by two 15-min incubations in dye-free buffer prior to sorting. Compared to our original prototype method in which Rh123, but not Ho, was effluxed, this dual-dye efflux protocol more rapidly and efficiently resolves the most primitive Hodull/Rhdull hematopoietic stem cells. Moreover, under conditions of optimal dual-dye uptake and efflux, Hodull/Rhdull cells map to the subfraction of side population (SP) cells with the highest efflux of Ho, which were previously demonstrated to possess the highest hematopoietic stem cell activity.
本文描述并展示了一种利用活体细胞染料Hoechst 33342（Ho）和罗丹明123（Rh123）的双染料外排策略，用于检测和分析小鼠骨髓中的造血干细胞。将骨髓细胞悬液中的单核细胞在Rh123加Ho的混合液中孵育，在分选前，通过在无染料缓冲液中进行两次15分钟的孵育使两种染料外排。与我们最初的原型方法（其中仅Rh123外排，Ho不外排）相比，这种双染料外排方案能更快速、有效地分离出最原始的Ho低表达/Rh低表达造血干细胞。此外，在最佳双染料摄取和外排条件下，Ho低表达/Rh低表达细胞定位于Ho外排最高的侧群（SP）细胞亚群，先前已证明该亚群具有最高的造血干细胞活性。
