BACKGROUND Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. METHODS Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction. RESULTS Flow cytometry analysis identified viable DRAQ5™+ /SYTOX® Blue- cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. CONCLUSIONS Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content. © 2018 International Clinical Cytometry Society.

中文翻译：

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基于DRAQ5™/SYTOX®Blue的新型流式细胞术策略，可识别和表征母乳中的干细胞。

背景技术人母乳可能是新生儿和早产儿发育的重要干细胞来源，但是需要各种妊娠阶段母乳中干细胞含量的定量数据，以确定作为干源的母乳的临床价值。细胞。母乳还包含乳脂球，不同大小的脂滴，碎片和死细胞，这些成分阻碍了人类母乳样品的流式细胞仪分析。方法在这里，我们最初使用标准协议进行流式细胞术来表征人母乳中的细胞群，但未能区分细胞和非细胞成分。然后，我们应用了离心程序从包含细胞的沉淀物中分离奶油和脱脂乳，并使用了带有DRAQ5™和SYTOX®蓝色染料以及用于表征沉淀部分中的细胞的抗体的新型染色方案。结果流式细胞仪分析鉴定出了人类母乳样品中可行的DRAQ5™+ /SYTOX®蓝细胞，并确定了CD11b +单核细胞和TRA-1-81 +推定干细胞的含量。结论因此，我们开发了一种新颖且可靠的基于流式细胞术的方法，用于量化人乳中具有高含量乳脂球，脂滴和颗粒的细胞亚群。这种方法将改善母乳细胞的鉴定和定量，并允许基于流式细胞仪的干细胞含量评估标准化。