Fluorescent imaging microscope

Exosome uptake by macrophages was examined as previously reported [8 (link)]. Differently treated OSCC cells were fixed, permeabilized and blocked as previously reported [15 (link)]. Then, the cells were incubated with anti-phospho-Stat3 (Y705) antibody (Cell Signaling Technology, USA) overnight at 4 °C followed by secondary antibody (Life, USA). Cellular nuclei were stained with DAPI (Roche, USA). Fluorescently labelled OSCC cells were examined using a ZEISS fluorescent imaging microscope (ZEISS, German).

HAECs were cultured in 4-well Lab-Tek chamber coated with collagen in Medium 200 plus Low Serum Supplement (Life Technologies) for 24 h. Exosomes from plasma were labeled with PKH67 (Sigma) for 30 min followed by PBS washes. HAECs were incubated with PKH67 labeled exosomes for 24 h. HAECs were fixed by 4% formaldehyde and the slide was mounted using ProLong Gold antifade reagent with DAPI (Life Technologies). The chamber slides and exosomes were visualized with a fluorescent imaging microscope (Zeiss).
In order to determiner the biological effects of JDM exosomes after their uptake by HAEC, we cultured HAECs in 6-well plates coated with collagen in Medium 200 plus Low Serum Supplement. When HAEC cells had approximate 85% confluence, they were incubated with exosomes from JDM (n = 4) or healthy control plasma (n = 4) for 24 h. HAEC total RNA was isolated using Trizol and RNeasy Mini Kit (Qiagen) as described previously [14 ].

The neurons seeded on coverslips were fixed with 4% paraformaldehyde in PBS, treated with 0.1% Triton X-100, and then were blocked by 5% bovine serum albumin. The samples were incubated with the mGluR1 primary antibody (#12551, Cell Signaling) at 4 °C overnight. After being washed by PBS with Tween-20 (PBST) for three times, the samples were incubated with the secondary antibody at 37 °C for 1 h. Then, DAPI (10 μg/ml) was used to stain the nuclei, and the pictures were obtained using a Zeiss fluorescent imaging microscope (Carl Zeiss, Thornwood, NY, USA).