Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 140, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/57528
Keywords
Immunology and Infection; Issue 140; Imaging Flow Cytometry; ImageJ-Fiji; IDEAS; Transcriptomics; Pathway analysis; Cytoscape
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Funding
- National Institute of Environmental Health Sciences, Center for Environmental Genetics (CEG) pilot project [ES006096]
- National Institute of Allergy and Infectious Diseases [AI115358]
- University of Cincinnati University Research Council award
- University of Cincinnati College of Medicine Core Enhancement Funding
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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 1 d 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 Mender'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.
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