Schneider s drosophila medium

Hemolymph was prepared from 30 flies at 12 days of age, as detailed under: “mRNA extraction” and was collected in the 1.5 mL Eppendorf tubes containing 50 µL of Schneider’s Drosophila Medium (Biological Industries, Beit-Haemek, Israel). Samples were centrifuged at 1100× g at 4 °C for 10 min, after which medium was discarded and the pellets were washed three times with cold PBS. Fifty microliters of Lysotracker^® Red DND-99 (Invitrogen, Eugene, OR, USA), diluted with Schneider’s Drosophila Medium to a final concentration of 1 µM, were added, followed by 15 min incubation at 25 °C, in the dark. Samples were washed three times with cold PBS and fixed with 4% paraformaldehyde/PBS (EMS, Hatfield, PA, USA) for 15 min at room temperature. Gallios flow cytometry (Beckman Coulter, CA, USA) was used to sort the hemocytes. LysoTracker signal was measured by side scatter using 530 nm excitation (SSC:FITC_530) and cell size was measured by forward light scatter (FSC:LinH). FlowJo software was applied for the analysis.

Drosophila Schneider S2R+ adherent cells were cultured in Schneider’s Drosophila Medium (Biological Industries) that was supplemented with 10% heat-inactivated fetal calf serum. Cells were transfected in 24-well plates by using the Escort IV reagent (Sigma-Aldrich). For dual luciferase assays, cells were plated at 6 x 10⁵ cells per each well of a 24-well plate one day prior to transfection. Each well was transfected with a total of 1 μg DNA composed of 930 ng of a vector control (mock -pAc empty), 60 ng of firefly luciferase Ftz and Ftz-F1 target gene reporter constructs and 2 ng of Scr-Renilla luciferase reporter. Co-activation experiments were performed by transfection of 25 ng of each expression vector (Ftz, Ftz-F1 or vector control). For the analyses of minimal core promoter constructs, no activator was added. Medium was replaced one day after transfection, and cells were harvested 36–48 hrs post transfection and assayed for dual luciferase activities, as specified by the manufacturer (Promega). To correct for variations in transfection efficiency, firefly luciferase activity of each sample was normalized to the corresponding Renilla luciferase activity. Each transfection was performed in triplicates, and each graph represents an average of at least 3 independent experiments.