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Browsing by Author "Makris, Melissa"

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    A flow cytometric method for measuring and isolating mammary epithelial cells from bovine milk
    Lengi, Andrea; Makris, Melissa; Corl, Benjamin A. (2021-11-01)
    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.
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    Heat Stress Increases Mammary Epithelial Cells and Reduces Viable Immune Cells in Milk of Dairy Cows
    Lengi, Andrea J.; Stewart, Jacob W.; Makris, Melissa; Rhoads, Michelle L.; Corl, Benjamin A. (MDPI, 2022-10-17)
    Somatic cells normally found in milk are generally either immune cells such as lymphocytes, monocytes and granulocytes, or mammary epithelial cells. The number and composition of somatic cells in milk can be influenced by a variety of factors, including infection and temperature-humidity index. The objective of this study was to determine the specific effects of heat stress on the cellular composition of the somatic cell population in milk. We used flow cytometry to ascertain the concentration and viability of mammary epithelial cells, T cells, monocyte/macrophage, and granulocytes in milk from cows maintained under heat stressed conditions compared to thermoneutral conditions. We found a significant 10% increase in the natural log concentration of epithelial cells in the milk of heat stressed cows compared to thermoneutral cows (9.3 vs. 8.4 ln(cells/mL, p = 0.02)). We also found a 12% decrease in the log concentration of live CD45+ cells (p = 0.04), and a 17% decrease in the log concentration of live CD45+ granulocytes (p = 0.04). No changes were found in CD3+CD45+ cells or CD14+CD45+ cells, however, we noted an unusual population of CD14+CD45− cells that showed significant increases of 10% (p = 0.03) and 12% (p = 0.01) in the log concentration of total and dead cells, respectively, under heat stressed conditions. These results suggest that heat stress influences the relative populations and viability of some somatic cells populations in milk. Increased losses of secretory epithelial cells into milk could have implications for milk production, and fewer viable immune cells could negatively impact the immunocompetence of dairy cows under heat stress.
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    Protoplast isolation prior to flow cytometry reveals clear patterns of endoreduplication in potato tubers, related species, and some starchy root crops
    Laimbeer, F. Parker E.; Holt, Sarah H.; Makris, Melissa; Hardigan, Michael Alan; Buell, C. Robin; Veilleux, Richard E. (Biomed Central, 2017-04-14)
    Background: Endoreduplication, the process of DNA replication in the absence of cell division, is associated with specialized cellular function and increased cell size. Genes controlling endoreduplication in tomato fruit have been shown to affect mature fruit size. An efficient method of estimating endoreduplication is required to study its role in plant organ development. Flow cytometry is often utilized to evaluate endoreduplication, yet some tissues and species, among them the tubers of Solanum tuberosum, remain intractable to routine tissue preparation for flow cytometry. We aimed to develop a method through the use of protoplast extraction preceding flow cytometry, specifically for the assessment of endoreduplication in potato tubers. Results: We present a method for appraising endoreduplication in potato (Solanum tuberosum) tuber tissues. We evaluated this method and observed consistent differences between pith and cortex of tubers and between different cultivars, but no apparent relationship with whole tuber size. Furthermore, we were able to observe distinct patterns of endoreduplication in 16 of 20 wild potato relatives, with mean endoreduplication index (EI) ranging from 0.94 to 2.62 endocycles per cell. The protocol was also applied to a panel of starchy root crop species and, while only two of five yielded reliable flow histograms, the two (sweet potato and turnip) exhibited substantially lower EIs than wild and cultivated potato accessions. Conclusions: The protocol reported herein has proven effective on tubers of a variety of potato cultivars and related species, as well as storage roots of other starchy crops. This method provides an important tool for the study of potato morphology and development while revealing natural variation for endoreduplication which may have agricultural relevance.