Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: CFP fluorescence was examined in whole mount fourth-instar larvae and pupae from PPO6 ( a ) or SPARC ( b ) transgenic lines. Arrowheads highlight presumed hemocytes. Scale bars: 1 mm. Potential differences in CFP expression between larvae and adult mosquitoes were examined by qPCR for both PPO6 ( c ) or SPARC ( d ) transgenic lines. For ( c and d ), expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled ( n = ~10) larvae or adult female mosquitoes, with values relative to rpS7 expression. Significance was determined using a two-tailed unpaired Student’s t -test. Exact P values are displayed in the figure where significant. ns not significant. Additional ex vivo analysis of perfused hemocytes from PPO6 ( e ) or SPARC ( f ) transgenic lines using microscopy was used to examine hemocyte fluorescence. The injection of fluorescent beads (red) prior to perfusion indicates that both PPO6-CFP + and SPARC-CFP + immune cell populations are comprised of phagocytic and non-phagocytic cells. Scale bars: 10 μm. Subpanels demonstrate different representative cell phenotypes. The specificity of CFP expression was examined in hemocytes and carcass samples via qPCR for the PPO6 ( g ) and SPARC ( h ) constructs across individual transgenic lines. The hemocyte-specific expression of NimB2 was used as a positive control for gene expression analysis. Expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled hemolymph perfusions ( n = >30 adult female mosquitoes) or adult female mosquitoes ( n = ~10) following perfusion (carcass), with values relative to rpS7 expression. Significance was determined using multiple two-tailed unpaired t -tests and a Holm–Šídák correction. Adjusted P values are displayed in the figure where significant. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Fluorescence, Transgenic Assay, Expressing, Two Tailed Test, Ex Vivo, Microscopy, Injection, Construct, Positive Control, Gene Expression

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: Flow cytometry analysis was performed on immune cell populations resulting from LRIM15 + , SPARC + , and PPO6 + individual transgenic lines ( a – f ). Representative UMAPs resulting from spectral imaging flow cytometry for LRIM15-GFP ( a ), SPARC-CFP ( c ), and PPO6-CFP transgenics ( e ) display the overall cell populations and the composition of fluorescent cells as an overlay using the established gating for each of the 12 hemocyte subpopulations identified in wild-type mosquitoes. The total number of events for each cell classification are displayed on the right of each figure subpanel. These distributions are summarized in pie charts to display fluorescent cell subtypes for LRIM15-GFP ( b ), SPARC-CFP ( d ), and PPO6-CFP transgenics ( f ). Data were averaged from three independent biological replicates. To determine potential overlap between transgenic markers, crosses were performed to establish either LRIM15 + /SPARC + ( g ) or LRIM15 + /PPO6 + ( h ) genetic backgrounds. For each genetic background, the abundance of GFP + , CFP + , and GFP + /CFP + cells were examined by overlaying fluorescent cell populations on the UMAP and summarized in bar graphs to denote the hemocyte clusters represented for each fluorescent cell subtype. Data display the average of three independent biological replicates. UMAP analysis was performed using the Euclidean distance metric using FlowJo V10.10.0, including the following parameters: DRAQ5 signal, Maximum Intensity Forward Scatter (FSC), Maximum Intensity Side Scatter (SSC), and Maximum Intensity Light Loss. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Flow Cytometry, Transgenic Assay, Imaging