The protocol was adapted from (Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). We set up large populations of flies in cages two days prior to ablation and placed a 35 or 60 mm grape juice agar plate (Lab Express, Ann Arbor, MI) in the cage with a dollop of goopy yeast. One day prior to the ablation, we replaced the grape juice/yeast plate with a new grape juice/yeast plate. On the morning of the ablation, we removed the plate from the cage and discarded the yeast puck and any hatched larvae on the agar. We then monitored the plate for up to four hours, until many larvae had hatched. Larvae were washed off the plate using a sucrose solution, and eggs were discarded. Larvae were then strained in coffee filters, and submerged in hydroxyurea (Sigma, H8627) in a yeast:AHL mixture, or sham mixture without HU. Ablation condition was 10 mg/mL HU, given for 1 hour. One batch experienced 15 mg/mL HU, given for 1 hour; this was done to gather more data points with a lower KC clone count. Larvae were then strained through coffee filters again, rinsed, and placed in a vial or bottle of B food (for MBON functional imaging and immunohistochemistry) or Janelia food supplemented with 0.2 mM all-trans-retinal (for behavior) until eclosion. We opened a new container of hydroxyurea each month as it degrades in contact with moisture, and we found its potency gradually declined. We achieved a U-shaped distribution of the HU effect, with many samples unaffected and many with all four KC neuroblasts lost. Ablated animals along with the control group (sham) were shipped in temperature-controlled conditions to Janelia Research Campus for behavior. A digital thermometer was kept in each shipment to record the lowest and highest temperature experienced during shipping. Batches that experienced ~15-27 Celsius were used for experiments. Animals were either shipped as larvae or late pupae, and not as adults in order to give enough time window for using the appropriate age of adult flies for behavior experiments.
Kenyon Cell Ablation in Drosophila
The protocol was adapted from (Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). We set up large populations of flies in cages two days prior to ablation and placed a 35 or 60 mm grape juice agar plate (Lab Express, Ann Arbor, MI) in the cage with a dollop of goopy yeast. One day prior to the ablation, we replaced the grape juice/yeast plate with a new grape juice/yeast plate. On the morning of the ablation, we removed the plate from the cage and discarded the yeast puck and any hatched larvae on the agar. We then monitored the plate for up to four hours, until many larvae had hatched. Larvae were washed off the plate using a sucrose solution, and eggs were discarded. Larvae were then strained in coffee filters, and submerged in hydroxyurea (Sigma, H8627) in a yeast:AHL mixture, or sham mixture without HU. Ablation condition was 10 mg/mL HU, given for 1 hour. One batch experienced 15 mg/mL HU, given for 1 hour; this was done to gather more data points with a lower KC clone count. Larvae were then strained through coffee filters again, rinsed, and placed in a vial or bottle of B food (for MBON functional imaging and immunohistochemistry) or Janelia food supplemented with 0.2 mM all-trans-retinal (for behavior) until eclosion. We opened a new container of hydroxyurea each month as it degrades in contact with moisture, and we found its potency gradually declined. We achieved a U-shaped distribution of the HU effect, with many samples unaffected and many with all four KC neuroblasts lost. Ablated animals along with the control group (sham) were shipped in temperature-controlled conditions to Janelia Research Campus for behavior. A digital thermometer was kept in each shipment to record the lowest and highest temperature experienced during shipping. Batches that experienced ~15-27 Celsius were used for experiments. Animals were either shipped as larvae or late pupae, and not as adults in order to give enough time window for using the appropriate age of adult flies for behavior experiments.
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Corresponding Organization : University of Michigan–Ann Arbor
Other organizations : Janelia Research Campus, Howard Hughes Medical Institute, Johns Hopkins Medicine, Johns Hopkins University
Variable analysis
- Hydroxyurea (HU) concentration: 10 mg/mL or 15 mg/mL
- Duration of HU treatment: 1 hour
- Number of Kenyon cell (KC) neuroblasts remaining (ranging from 0 to 4 per hemisphere)
- Sham treatment (without HU)
- Temperature during shipping (batches that experienced ~15-27 Celsius were used for experiments)
- Developmental stage of flies during shipping (larvae or late pupae, not adults)
- Positive control: Sham treatment (without HU)
- Negative control: Not explicitly mentioned
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