Drosophila Hemolymph Extraction and Proteomics

Embryos were collected from conventionally reared flies and dechorionated using a bleach-based previously published method [59 (link)] to ensure that they were devoid of horizontally transmitted pathogens. One generation was then left to develop in conventional rearing conditions to allow for gut microbiota recontamination. Hemolymph was extracted from 10 days-old flies from the second generation after bleach treatment using a Nanoject II (Drummond) as previously described [16 (link)]. 1 μL of hemolymph was collected for each sample and frozen at -80°C until further use. Hemolymph was then diluted 10 times with PBS containing Protease Inhibitor Cocktail 1X (Roche). 1 μl was used for protein quantification with the Pierce BCA Protein Assay Kit (Thermofisher). The remaining 9 μl were mixed with SDS (0.2% final), DTT (2.5mM final) and PMSF (10μM final). Aliquots of 15 μg were used for proteomics analysis.