The effects of zinc chelator TPEN in bovine mammary epithelial cells evaluated via a fluorescence resonance energy transfer approach

Document Type

Abstract

Publication Date

2020

Publisher

American Dairy Science Association

Journal

Journal of Dairy Science

Volume

103

Issue

Suppl. 1

Pages

235

Language

en

Keywords

zinc, cell culture, fluorescent protein

Abstract

Zinc plays important roles in many cellular functions. The free intracellular Zn is sensitive to chelating agents like TPEN (N,N,N’,N’-Tetrakis (2-pyridylmethyl) ethylenediamine). Therefore, we evaluated the effect of TPEN on changes in the intracellular Zn in bovine mammary epithelial alveolar cells (MacT). 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 a fluorescence resonance energy transfer (FRET) technology. Cells were seeded 24h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 at 0.3 µL/well and 150 ng/well of plasmid in reduced serum medium (OptiMEM). The transfected cells were treated in triplicates with 5 µM of TPEN (TPEN), 50 μM of Zn (Zn), and 50 μM of Zn + 5 µM 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 fluorescence resonance energy transfer (FRET) signal and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. A treatment × time effect (P < 0.01) was observed in FRET signal, where FRET was similar (P ≥ 0.18) across treatments at 0h but resulted in greater (P < 0.01) FRET in TPEN in comparison to Zn and Zn+TPEN at 24h. This suggests lower intracellular Zn concentration in TPEN than Zn and Zn+TPEN at 24h. The cell viability at 24h was similar (P ≥ 0.68) across treatments. These preliminary data indicate that TPEN is a viable model to study molecular alterations in Zn-depleted bovine cells and allow to evaluate additional conditions such as inflammation. FRET technology was sensitive to TPEN effects on intracellular Zn. To expand on these effects, gene expression and Zn concentration will be analyzed

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