In Vivo Phagocytosis Assay of Bacterial Particles

We performed the in vivo phagocytosis assay according to the previous method with some modification [48 (link)]. Alexa 594-labeled E. coli (K-12 strain) and Staphylococcus aureus (Wood strain without protein A) BioParticles conjugate (Invitrogen, Carlsbad, CA, USA) preparations (1 mg/mL) were injected into day 2 of the 5th instar larvae. Two hours later, hemolymph was collected from five larvae per diet and mixed with 1 μL of 1% PTU. One microliter of hemolymph was dropped onto a microslide which contained 100 μL Grace medium (Invitrogen). Slides were kept in the dark for one hour to adhere the hemocytes. The medium was removed and hemocytes were washed with PBS buffer three times, and then fixed with 4% paraformaldehyde for 15 min in the dark and washed three times with PBS buffer. The washed slides were covered with Antifade Mounting Medium (Solarbio, Beijing, China) and scanned using a fluorescent inverted microscope. Phagocytic index = mean fluorescence intensity of hemocytes in fluorescence-positive gate × (number of hemocytes in fluorescence-positive gate/total number of hemocytes). The percent of phagocytic hemocytes = phagocytic hemocytes/total number of hemocytes [49 (link)]. At least five views were observed per slide, and the assay was performed four times.