![Figure 4. EEV fractions promote the directional migration of human matured MMDCs along guidance cues in vitro and ex vivo. (a) Fluorescence images of Hoechst-stained (blue) MMDCs that were embedded in a 3D collagen matrix that contained SYTO RNASelect–stained TNFα-EEV fractions (green, left) or SYTO RNASelect–stained EEV-free supernatants (green, right; n = 5). Bars, 5 µm. (b) Flow cytometry analysis of MMDCs incubated for 60 min with PKH67-stained EEV-free supernatants or PKH67-stained TNFα-EEV fractions at 37°C or 4°C (n = 3; unpaired two-tailed t test). (c) Quantitation of MMDCs that directionally transmigrated through the porous membrane of the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded into the upper cell culture insert after the lower well chamber was loaded with EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions (n = 3; unpaired two-tailed t test). (d) Time-lapse image series (first frame top left image [i], last frame bottom right image [xii]) over 40 min of fluorescent MMDCs transmigrating a CCL21-overexpressing LEC monolayer in the presence of EEV-free supernatants (left), ss-EEV fractions (middle), or TNFα-EEV fractions (right). Arrows mark nontransmigrated MMDCs, and arrowheads mark transmigrated MMDCs (n ≥ 4). Bars, 15 µm. (e) Relative quantitation of MMDCs transmigrating a CCL21-overexpressing or -nonoverexpressing LEC monolayer in the presence of EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions (n ≥ 4; unpaired two-tailed t test with Welch’s correction). (f) Fluorescence images of a split-ear crawl-in assay. Fluorescently labeled MMDCs (color coded) were allowed to migrate for 3 h toward exposed dermal fluorescent lymphatic vessels (gray) in the presence of EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions. Color coding indicates the minimum distance of MMDCs to the nearest lymphatic vessel (n = 4). Bars, 200 µm. (g) Mean minimum distances between the nearest lymphatic vessels and MMDCs (described in f) in the presence of EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions were calculated and normalized to the overall vessel density (n = 4; unpaired two-tailed t test). Values represent means ± SEM. *, P ≤ 0.05; **, P ≤ 0.01.](https://cdn.rupress.org/rup/content_public/journal/jcb/217/6/10.1083_jcb.201612051/6/jcb_201612051_fig4.jpeg?Expires=1771570616&Signature=zxgHQ0XXsS8cvrkFIsxIdnsR~4uj2vLn7SdBztVqYrgjIboWSieUSPt5pOJkMaRgMJ8pzsQ6RVWFsCZI76C2-Zm3SSe479MmVonnQnsC9hLxAX8htkOh59ec7rQya44c~XYsmnn-VMwpNnWaADHaNZTQQEROmqE8Uv9MWRXD87PuWKKRPl~LMaZ4F13rGFqh~2CMmwbF9ruXfjVWgvwVB5eUEWDl256Nz4E~nbd0xpRMcZUzwTS7S~DHwUnexs~8s9EH016Jti8JpzFlCX7Mv21ZQbf699eEOpIIEIWu9rl1L7YWJiHaUERYqrQzx4Cp6W5hDzJiJbO5Lf3VhccHhA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
EEV fractions promote the directional migration of human matured MMDCs along guidance cues in vitro and ex vivo. (a) Fluorescence images of Hoechst-stained (blue) MMDCs that were embedded in a 3D collagen matrix that contained SYTO RNASelect–stained TNFα-EEV fractions (green, left) or SYTO RNASelect–stained EEV-free supernatants (green, right; n = 5). Bars, 5 µm. (b) Flow cytometry analysis of MMDCs incubated for 60 min with PKH67-stained EEV-free supernatants or PKH67-stained TNFα-EEV fractions at 37°C or 4°C (n = 3; unpaired two-tailed t test). (c) Quantitation of MMDCs that directionally transmigrated through the porous membrane of the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded into the upper cell culture insert after the lower well chamber was loaded with EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions (n = 3; unpaired two-tailed t test). (d) Time-lapse image series (first frame top left image [i], last frame bottom right image [xii]) over 40 min of fluorescent MMDCs transmigrating a CCL21-overexpressing LEC monolayer in the presence of EEV-free supernatants (left), ss-EEV fractions (middle), or TNFα-EEV fractions (right). Arrows mark nontransmigrated MMDCs, and arrowheads mark transmigrated MMDCs (n ≥ 4). Bars, 15 µm. (e) Relative quantitation of MMDCs transmigrating a CCL21-overexpressing or -nonoverexpressing LEC monolayer in the presence of EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions (n ≥ 4; unpaired two-tailed t test with Welch’s correction). (f) Fluorescence images of a split-ear crawl-in assay. Fluorescently labeled MMDCs (color coded) were allowed to migrate for 3 h toward exposed dermal fluorescent lymphatic vessels (gray) in the presence of EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions. Color coding indicates the minimum distance of MMDCs to the nearest lymphatic vessel (n = 4). Bars, 200 µm. (g) Mean minimum distances between the nearest lymphatic vessels and MMDCs (described in f) in the presence of EEV-free supernatants, ss-EEV fractions, or TNFα-EEV fractions were calculated and normalized to the overall vessel density (n = 4; unpaired two-tailed t test). Values represent means ± SEM. *, P ≤ 0.05; **, P ≤ 0.01.
