Journal: The Journal of Cell Biology
Article Title: Lymphatic exosomes promote dendritic cell migration along guidance cues
Figure Lengend Snippet: CX3CL1 in EEV fractions promotes transmigration of prostate carcinoma cells. (a) Immunofluorescence of CD9 (red) and podoplanin (green) in the peritumoral stroma of a human prostate carcinoma (fluorescence profiles across respective vessels are plotted above the merged images; x axis, cross-sectional distance; y axis, CD9- or podoplanin-specific mean immunofluorescence intensities; n = 5). Bars, 10 µm. (b) Flow cytometry histogram of unstained (red), APC-conjugated isotype IgG-stained (blue), and APC-conjugated anti-CX3CR1 IgG-stained (green) PC3-ML prostate carcinoma cells. X axis, percentage of maximum; Y axis, mean fluorescence intensity (MFI) of APC-conjugated IgG ( n = 4). (c) Microfluidic adhesion setup (top). EEV fractions were immobilized in microfluidic capillaries, and the adhesions of PC3-ML prostate carcinoma cells were acquired under constant-flow conditions for 2 min. Brightfield images of cancer cells (bottom) that adhered to EEV-free supernatant-coated (left), ss-EEV fraction-coated (middle), or TNFα-EEV fraction-coated (right) microfluidic channels ( n = 3). Bars, 50 µm. (d) Automated quantitation of adherent PC3-ML cell tracks in EEV-free supernatant-coated, ss-EEV fraction-coated, or TNFα-EEV fraction-coated ( n = 3; unpaired two-tailed t test) microfluidic channels. (e) Brightfield images of transmigrated PC3-ML prostate carcinoma cells from the upper cell culture insert into the lower chamber well of a transwell assay. PC3-ML cells were loaded into the upper cell culture inserts after EEV-free supernatants (leftmost), ss-EEV fractions (center left), TNFα-EEV fractions (middle), TNFα-EEV fractions plus anti-CX3CL1 IgG (center right), or TNFα-EEV fractions plus isotype control IgG (rightmost) were loaded into the bottom chamber wells ( n = 3). Bars, 100 µm. (f) Quantitation of transmigrated PC3-ML cells described in d ( n = 3; unpaired two-tailed t test). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.
Article Snippet: The antibodies against human β-actin, CD9, CD63, and CX3CL1 were purchased from Santa Cruz Biotechnology, Inc.
Techniques: Transmigration Assay, Immunofluorescence, Fluorescence, Flow Cytometry, Cytometry, Staining, Quantitation Assay, Two Tailed Test, Cell Culture, Transwell Assay