Gibco schneider s drosophila medium

In situ ROS detection was performed using dihydroethidium (DHE; D11347, ThermoFisher Scientific), as previously described [49 (link),115 (link)]. At ZT 0–2, flies were anesthetized on ice and whole guts were dissected in Gibco Schneider’s Drosophila Medium (21720024, ThermoFisher Scientific). The tissue was then incubated at room temperature with 60 μm DHE for 5 min in the dark. Next, tissues were washed 3x in Schneider’s medium for 5 min and then once in PBS for 5 min. Samples were then mounted in Vectashield with DAPI between a glass slide and coverslip and then imaged immediately on a Nikon A1R confocal microscope (Nikon) using a 10X air objective. Total ROS levels were quantified from pixel intensities of the Z-stack projection (sum slices). An ROI (gut tissue) was determined from the DAPI channel and then the mean of the summed DHE intensity averaged from each tissue was used for statistical analysis. Images are presented as the Z-stack projection through the entire gut and were processed using ImageJ2.

Embryos were collected and dechorionated as above. 100-200 embryos were placed in a micro-centrifuge tube containing 800 µl of GIBCO™ Schneider's Drosophila medium (Invitrogen) and dissociated using a sterile pestle. The suspension was centrifuged at 40 g for 5 min and the process repeated. The cell suspension was diluted to 1200 µl with medium. 300 µl of this was loaded into each chamber of a 4-well Lab-Tek™ II Chambered Coverglass (Nunc, Thermo Fisher Scientific, Rochester, NY, USA) pre-coated with Poly-L-Lysine (Sigma-Aldrich) for 1 h and washed with sterile water. Cells were loaded using 1 µM Fluo-4 (Invitrogen) for 1 h. Confocal microscopy was performed as described below. When required, thapsigargin (Sigma-Aldrich) was added to give a concentration of 2 µM. For real-time Ca²⁺ release experiments, cells were imaged with a 20× objective on a Zeiss LSM 710 (Carl Zeiss Ltd, Hertfordshire, UK) microscope using maximum scan speed without averaging.