TY  - JOUR
A1  - Ye, Mingyu
A1  - Keicher, Markus
A1  - Gentschev, Ivaylo
A1  - Szalay, Aladar A.
T1  - Efficient selection of recombinant fluorescent vaccinia virus strains and rapid virus titer determination by using a multi-well plate imaging system
JF  - Biomedicines
N2  - Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte\(^®\)S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.
KW  - fluorescent recombinant vaccinia virus
KW  - plaque isolation
KW  - IncuCyte\(^®\)S3
KW  - plaque assay
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245104
SN  - 2227-9059
VL  - 9
IS  - 8
ER  - 
TY  - JOUR
A1  - Ye, Mingyu
A1  - Wilhelm, Martina
A1  - Gentschev, Ivaylo
A1  - Szalay, Aladár
T1  - A modified limiting dilution method for monoclonal stable cell line selection using a real-time fluorescence imaging system: A practical workflow and advanced applications
JF  - Methods and Protocols
N2  - Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte\(^®\)S3.
KW  - monoclonal stable cell
KW  - limiting dilution cloning
KW  - ncuCyte\(^®\)S3
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228896
VL  - 4
IS  - 1
ER  -