Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking.

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander's coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.