Abstract
Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.
Highlights
Milk naturally contains somatic cells besides the well-known biochemical components, i.e. water, lactose, protein, fat, minerals
Cytometry gating strategy was developed by using blood cell samples stored at 4°C in phosphate buffer saline (PBS) and milk cell samples heattreated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively
The somatic cells were better preserved in milk after 72h storage, in particular polymorphonuclear neutrophils (PMNs), that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS)
Summary
Milk naturally contains somatic cells besides the well-known biochemical components, i.e. water, lactose, protein, fat, minerals. These milk somatic cells are made up of four main cell types: macrophages, polymorphonuclear neutrophils (PMNs) and lymphocytes that exist initially in blood and epithelial cells in the mammary glands. Flow cytometry is a favored method used to have information on the physiological status of somatic cells after milking. This accurate and reproducible method is routinely used to evaluate the total number of somatic cells present in milk of different species [4,5]. To quantify the cell viability, the exclusion markers i.e. propidium iodide, 7-Aminoactinomycin D, acridine orange or their combination are usually used to distinguish the viable and dead cells
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