Generation of Monoclonal Cultures from Wolbachia-infected Drosophila melanogaster JW18 Cell Line

We aim to generate monoclonal JW18 cell lines to study Wolbachia phenotypes in homogeneous cell populations. They should have consistent characteristics and produce less experimental variability than mixed-cell populations. Our protocol is comparatively low cost, avoids specialized equipment, minimizes cell stress, and enables reproducible generation of clonal insect cell cultures.

We will be able to follow evolution of Wolbachia bacteria in monoclonal cell culture and estimate the rate of Drosophila genome evolution in cell culture.

[Narrator] To begin, prepare fresh culture medium by supplementing Shield and Sang Insect Medium with 10% FBS. Using a 25 square centimeter flask, grow JW18 cells in fresh FBS-supplemented Shield and Sang Insect Medium at 25 degrees Celsius. Then scrape the cells from the flask, resuspend them in fresh medium, add cells to a new flask, and label it. Incubate the flask until the cells reach confluency approximately one week. Now, transfer the cell culture media from the flask to a 50-milliliter tube. Filter the one-week-old spent medium first through a five-micrometer sterile filter, followed by a 0.2-micrometer sterile filter fitted to a 50-milliliter syringe to remove Wolbachia, cell debris, and other contaminants. Now, with a serological pipette, pipette six milliliters of filtered spent medium and 24 milliliters of fresh medium to prepare conditioned medium. For the preparation of JW18 cell suspension, use a cell scraper to scrape the cells from a confluent one-week-old flask. Resuspend them using a serological pipette. Count the cells using a Neubauer chamber. Dilute the suspension with conditioned medium to a final concentration of 2,000 cells per milliliter. Transfer the conditioned medium to a reagent reservoir, then use a multi-channel pipette to add 100 microliters of conditioned medium to each well of a 96-well plate, except Well A-1. Now pipette 200 microliters of the cell suspension to Well A-1. Using a single-channel pipette, transfer 100 microliters from Well A-1 to Well B-1 and mix gently to avoid bubbles. Discard the final 100 microliters from the last well. Next, add 100 microliters of conditioned medium to each well in the first column to achieve a volume of 200 microliters and mix. With a multi-channel pipette, transfer 100 microliters from the first column to the second and mix gently. Repeat dilution for columns one to 12 across the plate. After mixing, discard 100 microliters from the last column, so each well contains 100 microliters. Add another 100 microliters of culture medium to bring the final volume to 200 microliters per well. Cover the plate, seal it with parafilm, and label it appropriately. Next, observe the plate under a microscope, scanning each well and its edges to confirm the presence of single cells. Record the well number of each single cell on a 96-well plate map on paper. After the identification of single cells, place the plate in an incubator set to 25 degrees Celsius. Inspect the wells every two to three days for bacterial contamination and to ensure the medium has not dried out. Top up with fresh medium, if needed. Once the cells reach confluence in the 96-well plate, transfer them to a 24-well plate. Subsequently, transfer the cells to a six-well plate, and finally to a flask. The first signs of cell division were observed on day three, with wells containing approximately four cells each. By day 14, clonal growth was observed either as a monolayer or as a sphere in JW18-G4. On day 28, visible differences in growth rates were most apparent among the clones. The JW18-C7 clone reached confluence by day 45, while the remaining clones achieved continuous growth only by day 77. By the 30th passage, the four established clones displayed distinct cell morphologies. PCR analysis detected Wolbachia wsp gene in all clones, except JW18-C7, indicating its loss of infection. FISH analysis confirmed Wolbachia presence in JW18-B9, JW18-E5, and JW18-G4, but not in JW18-C7.