Fig. 4.

PPO6^high and PPO6^low cell subpopulations share functional and morphological features. (A) Magnetic bead isolation assays followed by RNA-FISH for identification of cell populations. Arrows indicate lower PPO6 signal. (B) Intersection analyses between PPO6^high and PPO6^low cell populations and proteomics results obtained by Smith et al. (26) for phagocytes and all cells (unselected). The presence or absence of PPO genes/proteins in each of the samples is shown in B′. (C) Hemocytes were perfused from PPO6::RFP mosquitoes and analyzed using imaging flow cytometry. RFP-positive cells were identified by their RFP Median Pixel and further separated into PPO6^high and PPO6^low based on their level of RFP fluorescence. The number of cells analyzed per group is shown between parentheses, and the dotted line indicates the RFP threshold level used for separation of the two populations. (D) Image gallery containing representative images of PPO6^high and PPO6^low populations. Variation in cell shape and RFP intensity can be observed in the bright-field (BF), RFP, and merged images. (E) Box plots show the distribution of five morphological measurements according to the groups. Asterisks represent P < 0.01 based on Mann–Whitney–Wilcoxon test. (F) RFP-positive cells were interrogated for the presence of membrane protrusions or internal structures suggestive of vesicles. Similar to C, cells were grouped into PPO6^high and PPO6^low, and an RFP intensity histogram of cells displaying vesicles (green population) was generated to illustrate that vesicles are observed in cells from both groups. Representative images of cells in this population subset are shown in G. Arrow and arrowhead indicate representative membrane protrusions and internal vesicles, respectively. Box plots indicate the median, first and third quartile, and min and max values. (Magnification: D and G, 40×.)