Intracellular zinc bioavailability in bovine mammary epithelial cells is modulated by lipopolysaccharide: A fluorescence resonance energy transfer approach
2019 American Dairy Science Association Annual Meeting: Cincinnati, Ohio
American Dairy Science Association
Journal of Dairy Science
zinc, bovine cells, lipopolysaccharide (LPS)
Zinc is an important trace element which is involved in many intracellular pathways including cell growth, differentiation, and inflammation. Therefore, we evaluated the effect of the intracellular bioavailability of Zn in bovine mammary epithelial cells (MacT) in response to an LPS 287J. Dairy Sci. Vol. 102, Suppl. 1 (Escherichia coli O111:B4, Sigma-Aldrich) challenge. To test this, MacT cells were seeded 24h before transfection at 30,000 cells/well in a 96-well plate. Additional cells were seeded at 300,000 cells/well in a 6-well plate for gene expression analysis. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect Zn through fluorescence resonance energy transfer (FRET) technology. Cells were transfected with the transfection reagent Lipofectamine 3000 at 0.3 μL/ well and at 150 ng/well of plasmid in a reduced serum medium deprived of FBS. Transfected cells were treated with 50 μM concentration of Zn, and challenged at non-clinical (0 µg of LPS/mL) and clinical levels (250 µg of LPS/mL) for 2 h at 0, 6, and 12 h after the treatment. An inverted fluorescent microscope for live imagining equipped with scanning stage, and an environment-controlled chamber at 37°C and 5.0% of CO2 was used to take 4 pictures/well at 4× magnification. Quantification of Zn and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. Overall intracellular availability of Zn was not affected by LPS in MacT cells incubated with 50 µM Zn at 0 and 12 h post-treatment. However, at 6 h post Zn treatment, there was a significant increase (P < 0.01) in intracellular bioavailability of Zn compared with the control over time during LPS challenge. Within 1 h post LPS challenge, the Zn intracellular bioavailability was greater (P = 0.05) in the challenged cells than the control. Similarly, at 1.5 and 2 h post LPS challenge, the Zn intracellular bioavailability was greater (P < 0.001) in the challenged cells than the control. These preliminary data indicate that FRET technology is sensitive enough to detect changes in intracellular Zn content mediate through an LPS model. To expand on these effects, gene expression analysis will be performed.
Mohan, R.; Rosa, F.; and Osorio, J. S., "Intracellular zinc bioavailability in bovine mammary epithelial cells is modulated by lipopolysaccharide: A fluorescence resonance energy transfer approach" (2019). Dairy Science Publication Database. 2139.