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A fluorescence resonance energy transfer approach to determine intracellular bioavailability of zinc in blood samples using a bovine mammary epithelial cell model.

R. Mohan

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06-23-2020

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Abstract:

T62
A fluorescence resonance energy transfer approach to determine intracellular bioavailability of zinc in blood samples using a bovine mammary epithelial cell model.
R. Mohan*, F. Rosa, J. S. Osorio. Dairy and Food Science Department, South Dakota State University Brookings, SD.

This study evaluated the intracellular bioavailability of Zn in blood plasma and serum from dairy cows using a Zn fluorescent resonance energy transfer (FRET) system. Bovine mammary alveolar epithelial cells (MacT) were incubated with either serum or plasma at 5, 10, 25, 50, 75, and 100% diluted with reduced serum medium (OptiMEM). Before transfection, MacT cells were cultivated in high glucose Dulbecco modified Eagle's medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and Fungizone Antimycotic. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect intracellular Zn through FRET technology. Cells were seeded 24 h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 at 0.3 uL/well and at 150 ng/well of plasmid in OptiMEM. Free intracellular Zn can be depleted by chelating agents such as TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine). Hence, additional treatments included 5 �M of TPEN(TPEN), 50μM of Zn (Zn), and 50μM of Zn+5 �M of TPEN (Zn+TPEN). An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with an environment-controlled chamber at 37�C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification at 0 and 24 h post-treatment. Quantification of FRET signal and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. A treatment effect (P < 0.01) in FRET signal was observed in serum and a trend (P = 0.11) in plasma samples. There was an evident decline in FRET signal as serum % was increased with lower (P < 0.01) FRET at 100% serum, this suggests an increase in intracellular Zn. There were no differences in FRET across plasma %. The cell viability at 24h was similar across serum (P ≥ 0.54) and plasma (P ≥ 0.34) treatments. These preliminary data indicate that the available Zn in serum can be detected via FRET in bovine cells. To expand on these effects, gene expression and Zn concentration will be analyzed.

Keywords: zinc, cell culture, fluorescent protein.

Biography: I am presently pursuing PhD in Dairy Production at Department of Dairy and Food Science, South Dakota State University, USA. My research is mainly on 'Advanced fluorescent protein systems for dairy cattle nutrigenomics.