A flow cytometric method for measuring and isolating mammary epithelial cells from bovine milk

Sampling frequent timepoints 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 the 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 kappa casein mRNA expression in BTN single positive cells compared to unsorted cells, while 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 kappa casein mRNA expression compared to unsorted cells. We found no enrichment of kappa casein mRNA expression after sorting cells using cytokeratin antibodies. The non-invasive assays described here can allow for daily or more frequent sampling timepoints for measurement of mammary epithelial cells during the course of lactation.