Bx51 fluorescent microscopy

The MARCM tool yw, hs-Flp, tub-Gal4, UAS-mCD8::GFP; tub-Gal80, FRT40A/CyO was used to generate GFP positive MARCM clones [24 (link)]. To perform the screen, the virgins of yw, hs-Flp, tub-Gal4, UAS-mCD8::GFP; tub-Gal80, FRT40A/CyO were collected and mated to FRT-P-element lethal lines. Then the 2^nd and 3^rd instar larvae from these crosses were heat-shocked at 37°C for two hours every day in three days. After eclosion, non-curly females were fed with fresh yeast for 2–3 days. Ovaries were dissected in PBS and mounted in medium. For BC migration examination, fluorescent images of stage 10 egg chambers was taken using Olympus BX51 fluorescent microscopy. According to the position of BC clusters at stage 10, each egg chamber was classified into two classes, Normal or Delay. The Normal class was identified when BC cluster reached the border between nurse cells and the oocyte at stage 10. The Delay class was identified when BC cluster did not reach the border and stayed among nurse cells at stage 10.

After growing them at 40.5 °C for 2.5 h, cells were harvested by 3000 rpm for 5 min, washed with spheroplast buffer [1.2 M sorbitol, 10 mM K-PO₄ (pH7.5)] and suspended in 3 mL spheroplast buffer containing 1 mg/mL zymolyase 100 T (Nacalai tesque, 07665-55). Cells were incubated at 40.5 °C for 30 min, centrifuged at 3000 rpm for 5 min, and suspended in 100 μl of lysis buffer (0.2 M sorbitol, 10 mM K-PO₄ (pH7.5)). When cells grown at 25 °C were examined, a zymolyase treatment was performed at 30 °C for 1 h. Fast Dil (Thermo Fisher D7756) was added at the final concentration of 5 μg mL⁻¹ from the stock solution (1 mg mL⁻¹ DMSO), and vacuoles contained in the lysate were imaged with the Olympus BX51 fluorescent microscopy. Images were processed by GIMP or Photoshop.