Immunofluorescence Microscopy and Flow Cytometry Characterization of Chemical Induction of Latent Epstein-Barr Virus

Abstract

The effects of chemical induction of latent Epstein-Barr virus (EBV) with 12-O-tetradecanoyl phorbol-13-acetate (TPA) andn-butyrate on cell viability and induction of latent EBV in Raji and X50-7 B lymphocytes, indicated by expression of the diffuse component of the EBV early antigen (EA-D), were measured by visual immunofluorescence microscopy (of both viable and nonviable cells) and fluorescence-activated cell sorter (FACS) flow cytometry (of viable cells only). Cell viability at 4 days decreased moderately for treated Raji cells (9 to 37%, compared to 55 to 69% for untreated cells) and markedly for X50-7 cells (1-32% compared to 35-44% in untreated cells). The highest EA-D levels in viable cells occurred in Raji cells treated with both TPA and n-butyrate and untreated X50-7 cells. TPA and n-butyrate acted synergistically to induce latent EBV, resulting in increased levels of EA-D production in Raji cells and cell death in X50-7 cells. Methodological differences including the ability to detect antigen in only viable cells by FACS flow cytometry accounted for the higher levels of EA-D observed by FACS analysis compared to the levels observed by immunofluorescence microscopy. FACS analysis may be more objective and reproducible than immunofluorescence microscopy for the detection of EBV induction and also permits viral protein expression to be distinguished in the subpopulation of viable cells.

Mechanosensitive mTORC1 signaling maintains lymphatic valves

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Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

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Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

“…These results are in contrast to those reported by other authors (Anzar & Buhr, 2006;Canovas et al, 2010), who describe that only a portion of sperm bind exogenous DNA after co-incubation (49 and 29%, respectively). The differences observed could be a result of the assessment method used since it has been described that the detection of proteins by flow cytometry, the technique used in the present study, can be at least 10 times more sensitive than detection by conventional fluorescence microscopy (Bolanos et al, 1988;Jenson et al, 1998;Soboleski et al, 2005). Alternatively, the differences could be related to the different components present in the media since it has been described that media with Ca 2+ and BSA accelerate the sperm capacitation processes (Visconti et al, 1999;Aguila et al, 2015), which is unfavourable for binding exogenous DNA by sperm, which occurs in the early stages of capacitation (Lavitrano et al, 2003).…”