TY - JOUR A1 - Hornych, Ondrej A1 - Ekrt, Libor A1 - Riedel, Felix A1 - Koutecky, Petr A1 - Košnar, Jiří T1 - Asymmetric hybridization in Central European populations of the Dryopteris carthusiana group JF - America Journal of Botany N2 - Premise Hybridization is a key process in plant speciation. Despite its importance, there is no detailed study of hybridization rates in fern populations. A proper estimate of hybridization rates is needed to understand factors regulating hybridization. Methods We studied hybridization in the European Dryopteris carthusiana group, represented by one diploid and two tetraploid species and their hybrids. We sampled 100 individuals per population in 40 mixed populations of the D. carthusiana group across Europe. All plants were identified by measuring genome size (DAPI staining) using flow cytometry. To determine the maternal parentage of hybrids, we sequenced the chloroplast region trnL-trnF of all taxa involved. Results We found hybrids in 85% of populations. Triploid D. xambroseae occurred in every population that included both parent species and is most abundant when the parent species are equally abundant. By contrast, tetraploid D. xdeweveri was rare (15 individuals total) and triploid D. xsarvelae was absent. The parentage of hybrid taxa is asymmetric. Despite expectations from previous studies, tetraploid D. dilatata is the predominant male parent of its triploid hybrid. Conclusions This is a thorough investigation of hybridization rates in natural populations of ferns. Hybridization rates differ greatly even among closely related fern taxa. In contrast to angiosperms, our data suggest that hybridization rates are highest in balanced parent populations and support the notion that some ferns possess very weak barriers to hybridization. Our results from sequencing cpDNA challenge established notions about the correlation of ploidy level and mating tendencies. KW - antheridiogens KW - Dryopteridaceae KW - ferns KW - flow cytometry KW - hybridization rate KW - interspecific hybridization KW - polyploidy KW - reproductive isolation KW - speciation KW - trnL-trnF Y1 - 2019 U6 - https://doi.org/10.1002/ajb2.1369 SN - 0002-9122 SN - 1537-2197 VL - 106 IS - 11 SP - 1477 EP - 1486 PB - Wiley CY - Hoboken ER - TY - THES A1 - Dippong, Martin T1 - Direkte und indirekte Hapten-selektive Immunfluoreszenzmarkierung von Hybridomzellen zur Generierung monoklonaler Antikörper T1 - Direct and indirect hapten-specific immunofluorescence labeling of hybridoma cells for the generation of monoclonal antibodies N2 - Die Hybridomtechnik zur Produktion von monoklonalen Antikörpern ermöglichte einen großen Schritt in der Entwicklung von Immunoassays für die biochemische Forschung und klinische Diagnostik. Auch die Produktion von Antikörpern gegen niedermolekulare Analyten, Haptene, typische Targets in der Lebensmittel- und Umweltanalytik, erlangte in den letzten Jahren eine immer größere Bedeutung. Im Zuge der Durchführung der Hybridomtechnik werden tausende Antikörper-sezernierende und nicht-sezernierende Zellen generiert. Die Selektion der wenigen antigenselektiven Hybridomzellen zählt dabei zu den herausforderndsten Schritten für die Antikörpergewinnung. Bisherige Selektionsverfahren, wie die Limiting-Dilution-Klonierung in Verbindung mit Enzyme-linked Immunosorbent Assays (ELISAs), garantieren keine Monoklonalität und erlauben nur das Screening von einigen wenigen Zellklonen. Hingegen ermöglichen Hochdurchsatz-Selektionsmethoden, wie die Fluoreszenz-aktivierte Zellsortierung (FACS), einen sehr hohen Probendurchsatz. Eine Einzelzellablage garantiert hierbei Monoklonalität. Jedoch sind die dafür erforderlichen Zellmarkierungen oftmals zellschädigend oder aufwendig zu generieren. Auch ist bisher noch keine Markierungsmethode bekannt, die es ermöglicht, Hapten-selektive Hybridomzellen durchflusszytometrisch zu analysieren und eine FACS-Selektion durchzuführen. Aus diesem Grund wurden in dieser Arbeit zwei Zellmarkierungsmethoden entwickelt, die dies ermöglichen sollten. Die membranständigen Antikörper von Hybridomzellen sollten entweder direkt oder indirekt immunfluoreszenz-markiert und dadurch für die Durchflusszytometrie und FACS-Selektion zugänglich gemacht werden. Die direkte Markierung wurde mittels eines Hapten-Fluorophor-Konjugats durchgeführt. Sie ermöglichte erstmalig den Anteil an Haptenselektiven Hybridomzellen in einer Hybridomzelllinie zu überprüfen. Dies konnte für zwei Hapten-selektive Hybridomzelllinien, die Antikörper gegen das Hormon 17β-Estradiol und das Cardenolid Digoxigenin bilden, gezeigt werden. Durchflusszytometrie und ELISAs lieferten vergleichbare Ergebnisse. Zellen, die Hapten-selektiv markiert werden konnten, sezernierten ebenfalls Hapten-selektive Antikörper. Des Weiteren konnte die direkte Markierung dazu genutzt werden, zwei Mykotoxin-selektive Hybridomzelllinien, welche Antikörper gegen Aflatoxin und Zearalenon bilden, auf Monoklonalität zu testen. Dies ist mittels ELISA nicht möglich. Die Markierungsmethode eignete sich jedoch nur für fixierte Hybridomzellen. Eine Markierung von lebenden Zellen konnte weder durchflusszytometrisch noch mittels konfokaler Laser-Scanning-Mikroskopie gezeigt werden. Dies gelang erst mit einer neu entwickelten indirekten Immunfluoreszenzmarkierung. Dabei wurden die Zellen zunächst mit einem Hapten-Peroxidase-Konjugat inkubiert, gefolgt von einem Fluorophor-markierten anti-HRP-Antikörper-Konjugat. Dies wurde für zwei Analyten, das Hormon Estron und das Antiepileptikum Carbamazepin, gezeigt. Die indirekte Markierung wurde erfolgreich dazu verwendet, Carbamazepin-selektive Hybridomzellen aus einem Fusionsansatz für die monoklonale Antikörperproduktion auszusortieren. Damit wurde erstmalig eine Zellmarkierungsmethode entwickelt, die eine Hochdurchsatz-Selektion lebender Hybridomzellen aus einem Fusionsansatz ermöglicht. Sie ist nicht zellschädigend und kann zusätzlich zur Selektion Hapten-selektiver Plasmazellen verwendet werden. N2 - The ability to create monoclonal antibodies has allowed great strides to be made in immunoassay development for biochemical research and clinical diagnostics. Particularly for small molecular weight analytes, haptens, the need of selective antibodies has increased. The hybridoma technique generates thousands of fused antibody-secreting and non-secreting cells, with the majority being irrelevant. The subsequent screening and subcloning process in order to identify and isolate the very few hybrids that are secreting antibodies of the desired selectivity is a major concern. The traditional limiting dilution technique followed by enzymelinked immunosorbent assays (ELISAs) is inefficient and monoclonality is not guaranteed. Often the number of clones that can be screened is limited. High-throughput techniques such as fluorescence-activated cell sorting (FACS) provide an efficient tool to increase the number of cells to be screened. Furthermore, a single-cell deposition of cells would ensure monoclonality. However, antigen-selective cell labeling techniques are often cell damaging or laborious. The purpose of this study was to explore a cell labeling technique enabling the hapten-selective analysis and isolation of hybridoma cells via FACS. This would reduce much of the effort that has currently to be employed in hybridoma generation. For this reason, a direct and indirect hapten-selective labeling technique was developed. For the direct labeling, a haptenfluorophore conjugate was generated. The conjugate was used to tag membrane-bound immunoglobulin G of hybridoma cells and thereby enabling flow cytometric analysis. Using this kind of conjugate, it was possible to examine the selective antibody expression of hybridoma cell lines producing antibodies against the hormone estradiol and the steroid digoxigenin. Flow cytometric analysis and ELISAs showed comparable results: Cells, which were tagged with the corresponding hapten-fluorophore conjugate also secreted hapten-selective antibodies. Furthermore, it was possible to check hybridoma cell lines producing antibodies against the mycotoxins aflatoxin and zearalenone for monoclonality, which is not possible with ELISA. However, the direct labeling technique was only applicable to fixed cells. Successful labeling of living cells could neither be detected by flow cytometry nor by confocal laser scanning microscopy. On the contrary, using the newly developed indirect labeling technique, flow cytometric analysis and selection of living cells by FACS was possible. Here, the cells were first incubated with a hapten-peroxidase conjugate followed by a fluorophore-conjugated anti-peroxidase antibody. The technique was established on a hybridoma cell line selective for the hormone estrone. Furthermore, this labeling technique enabled for the first time the sorting of hybridoma cells producing selective antibodies against the medication carbamazepine out of a fusion mixture with high efficiency. The selected clones were used for monoclonal antibody production. The indirect labeling is harmless for cells and could also be applied on haptenselective plasma cells. KW - Durchflusszytometrie KW - Haptene KW - monoklonale Antikörper KW - Hybridom KW - Immunfluoreszenz KW - flow cytometry KW - hapten KW - monoclonal antibodies KW - hybridoma KW - immunofluorescence Y1 - 2017 ER - TY - JOUR A1 - Beisner, Beatrix E. A1 - Grossart, Hans-Peter A1 - Gasol, Josep M. T1 - A guide to methods for estimating phago-mixotrophy in nanophytoplankton JF - Journal of plankton research N2 - Growing attention to phytoplankton mixotrophy as a trophic strategy has led to significant revisions of traditional pelagic food web models and ecosystem functioning. Although some empirical estimates of mixotrophy do exist, a much broader set of in situ measurements are required to (i) identify which organisms are acting as mixotrophs in real time and to (ii) assess the contribution of their heterotrophy to biogeochemical cycling. Estimates are needed through time and across space to evaluate which environmental conditions or habitats favour mixotrophy: conditions still largely unknown. We review methodologies currently available to plankton ecologists to undertake estimates of plankton mixotrophy, in particular nanophytoplankton phago-mixotrophy. Methods are based largely on fluorescent or isotopic tracers, but also take advantage of genomics to identify phylotypes and function. We also suggest novel methods on the cusp of use for phago-mixotrophy assessment, including single-cell measurements improving our capacity to estimate mixotrophic activity and rates in wild plankton communities down to the single-cell level. Future methods will benefit from advances in nanotechnology, micromanipulation and microscopy combined with stable isotope and genomic methodologies. Improved estimates of mixotrophy will enable more reliable models to predict changes in food web structure and biogeochemical flows in a rapidly changing world. KW - flow cytometry KW - phagotrophy KW - phytoplankton KW - methods KW - fluorescence KW - microscopy KW - FISH KW - isotopic methods KW - phylotypes KW - carbon flows KW - gene sequencing Y1 - 2019 U6 - https://doi.org/10.1093/plankt/fbz008 SN - 0142-7873 SN - 1464-3774 VL - 41 IS - 2 SP - 77 EP - 89 PB - Oxford Univ. Press CY - Oxford ER -