Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

PLOS ONE RESEARCH ARTICLE Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes Mira Woitok1,2, Elena Grieger1, Olusiji A. Akinrinmade3, Susanne Bethke1,2, Anh Tuan Pham1, Christoph Stein1, Rolf Fendel1,4, Rainer Fischer1,2, Stefan Barth1,3, Judith Niesen ID1,5,6,7* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany, 2 Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany, 3 Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, University of Cape Town, Observatory, Cape Town, South Africa, 4 Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany, 5 Department of Pediatric Hematology and Oncology, University Medical Centre HamburgEppendorf, Hamburg, Germany, 6 Mildred Scheel Cancer Career Center HaTriCS4, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 7 Research Institute Children’s Cancer Center, Hamburg, Germany * OPEN ACCESS Citation: Woitok M, Grieger E, Akinrinmade OA, Bethke S, Pham AT, Stein C, et al. (2020) Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes. PLoS ONE 15(12): e0243286. https://doi.org/10.1371/journal. pone.0243286 Editor: Irina V. Lebedeva, Columbia University, UNITED STATES Received: April 17, 2020 Accepted: November 18, 2020 Published: December 3, 2020 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the manuscript and its Supporting information files. Funding: Mira Woitok was supported by the RWTH Aachen University scholarship of Young Researchers at the RWTH Aachen University (RFwN). This work was supported by the Fördergemeinschaft Kinderkrebs-Zentrum Hamburg, the Erich and Gertrud Roggenbuck Abstract In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research. Introduction Programmed cell death, or apoptosis, is a natural physiologic process during cell development of aging or homeostasis of cells. It may also facilitate the removal of unwanted, e.g. damaged, PLOS ONE | https://doi.org/10.1371/journal.pone.0243286 December 3, 2020 1 / 16 PLOS ONE Stiftung and the German Cancer Aid (via the Mildred Scheel Cancer Career Center). Competing interests: The authors have declared that no competing interests exist. Using the SNAP-Tag technology to easily demonstrate apoptotic changes in different cell types cells, which may result from e.g. different immune reactions [1]. In cancer, dysregulated cell death is also common and hence used as effective therapeutic line of attack [2–4]. Apoptosis is demonstrated by typical morphological changes such as cell shrinkage, packed organelles and an increased density of the cytoplasm. This results in a reduction in cell volume and the typical forming of apoptotic bodies, called budding. Additionally, chromatin condensation and nuclear fragmentation can be observed in cells undergoing apoptosis [1, 3, 5]. At the beginning of the nineties, it was found that annexin V (AV) bound calcium-dependently to phospholipid bilayers. In addition, it was discovered that phosphatidylserine (PS), a phospholipid located on the inner leaflet of the cell membrane in normal cells, is exposed on the surface of cells in the early apoptotic phase, for the specific recognition by lymphocytes, in this case macrophages [6, 7]. Since AV binds specifically, calcium-dependently and with high affinity to PS, it was employed for monitoring apoptotic cells, for example in flow cytometry assays [8, 9]. Also in the early nineties, the first flow cytometry assay using AV conjugated to FITC was evaluated, showing that fluorophore-conjugated AV can be used to detect apoptotic changes in cells with this method/assay [10]. In addition to apoptosis, cell death may alternatively occur by necrosis. In this case, the cells act passively since necrosis represents cell death that is triggered by external factors, diseases, infections, or toxins [1, 11]. The mechanisms and morphologic characteristics of cells undergoing necrosis are cell swelling, an expansion of the endoplasmic reticulum, the formation of cytoplasmic vacuoles as well as blebs, swelling of lysosomes and possibly disruption of the cell membrane [1]. To distinguish early apoptotic cells from late apoptotic and necrotic cells, propidium iodide (PI) is used in many flow cytometry assays. PI intercalates into DNA and can or may bind the nucleus of these late apoptotic/necrotic cells whose cell membrane is disintegrated [8, 12]. For these reasons, flow cytometry based AV/PI assays are commonly used to measure apoptotic and necrotic changes in target cells, such as cancer cells, which are exemplarily treated with novel therapeutic agents [8, 13–17]. Fluorescence activated cell sorting (FACS) based AV/PI-assays can also be combined with other methods such as staining approaches to detect cell death signaling pathways or differences in cell morphology [2]. However, this has drawbacks, such as that the fluorophore-conjugated AV must be exchanged if the fluorophore irradiates with other dyes used (...truncated)