Generation of Monoclonal Cultures from <em>Wolbachia</em>-infected <em>Drosophila melanogaster</em> JW18 Cell Line

Cell lines are widely used models in biological research. They are particularly useful in studies of intracellular bacteria that cannot be efficiently propagated outside host organisms. Wolbachia, an intracellular symbiont of many invertebrates, induces strong reproductive and antiviral effects in its insect hosts. Wolbachia-infected insect cells have been widely used to study Wolbachia phenotypes. However, these cell lines often consist of mixed populations of cells with potentially varying phenotypes and responses to experimental treatments. In particular, cell lines with Wolbachia have been reported to exhibit variable growth rates and variable Wolbachia infection prevalence from one passage to the next. To remedy this, we generated monoclonal cell lines from the Wolbachia-infected Drosophila melanogaster-derived JW18 cell line. These clonal lines were established at different timepoints and show different Wolbachia infection statuses. This variability suggests that any treatment applied to a parental JW18 population could lead to the selection of sub-populations as opposed to influencing the physiology of the entire culture. Here, we present a protocol for generating single-cell clones and continuous clonal cultures from Wolbachia-infected insect cells, enabling more controlled and reproducible experiments.