Lengi A J, Makris M, Corl B A
Department of Dairy Science, Virginia Tech, Blacksburg 24061-0315.
Flow Cytometry Laboratory, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg 24061.
JDS Commun. 2021 Aug 26;2(6):426-430. doi: 10.3168/jdsc.2021-0135. eCollection 2021 Nov.
Sampling frequent time points of mammary signaling pathways is not possible with tissue biopsies. We have validated a flow cytometry and cell sorting procedure for isolating live bovine mammary epithelial cells from somatic cell populations in milk using butyrophilin 1A1 as a marker for mammary epithelial cells and CD45 as a marker for hematopoietic cells. Hoechst 33342 staining and propidium iodide exclusion were used to select for nucleated live cells. Positive selection of butyrophilin (BTN)-expressing cells was performed by fluorescence-activated cell sorting. Quantitative real-time PCR performed on mRNA isolated from these cells showed a 226-fold increase in κ-casein () mRNA expression in BTN single-positive cells compared with unsorted cells, whereas CD45 single-positive cells showed a significant decrease. A negative selection strategy for cells not expressing the hematopoietic cell marker CD45 also resulted in a cell population with a 196-fold increase in mRNA expression compared with unsorted cells. We found no enrichment of mRNA expression after sorting cells using cytokeratin antibodies. The noninvasive assays described here allow for daily or more frequent sampling time points for measurement of mammary epithelial cells during the course of lactation.
通过组织活检无法对乳腺信号通路的频繁时间点进行采样。我们已经验证了一种流式细胞术和细胞分选程序，该程序使用嗜乳脂蛋白1A1作为乳腺上皮细胞的标志物，CD45作为造血细胞的标志物，从牛奶中的体细胞群体中分离活的牛乳腺上皮细胞。使用Hoechst 33342染色和碘化丙啶排除法来选择有核活细胞。通过荧光激活细胞分选对表达嗜乳脂蛋白（BTN）的细胞进行阳性选择。对从这些细胞中分离的mRNA进行的定量实时PCR显示，与未分选的细胞相比，BTN单阳性细胞中κ-酪蛋白（）mRNA表达增加了226倍，而CD45单阳性细胞则显著下降。对不表达造血细胞标志物CD45的细胞采用阴性选择策略，也得到了一个与未分选细胞相比κ-酪蛋白mRNA表达增加196倍的细胞群体。我们发现使用细胞角蛋白抗体分选细胞后，κ-酪蛋白mRNA表达没有富集。本文所述的非侵入性检测方法允许在泌乳过程中每天或更频繁地对乳腺上皮细胞进行采样时间点测量。
