Measuring Hemocyte Responses to Parasitic Infections

The msn-mCherry D. melanogaster strain was used to assay expression of msn. This strain carries a transgenic construct containing the msn-F9 enhancer upstream of the mCherry red fluorescent protein [51 (link)]. Second instar msn-mCherry larvae were exposed to either AsDen or L. boulardi for a 72-h period as described above, with 3 biological replicates for each infection condition. Host hemocytes were isolated 72 hpi and added to a Tali Cellular Analysis Slide (Invitrogen). Hemocytes were allowed to adhere for 30 min and then cell number, size, perimeter, circularity, and red fluorescence intensity were measured using a Tali Image-Based Cytometer (Invitrogen). For each replicate, we imaged 20 fields of cells, with an average of 717.4 cells per field, and a range of 194 to 1455 cells for a total of 32,176 hemocytes from L. boulardi-infected larvae and 53,908 hemocytes from AsDen-infected larvae. Cytometer data were filtered to only include single cells using the Tali software count function and size-gating, prior to further analysis.