Anti mouse alexa fluor 647

A1 cells at ~ 80% confluency were transfected with a plasmid that generates a fusion protein of Green Fluorescent protein (GFP) and the tetraspanin protein, CD63, under the control of the CMV promoter (pCT-CD63-GFP, System Biosciences # CYTO120-PA-1). 2.5 μg of plasmid in 200 μL of serum-free, dilute MEM (70%) was mixed with 6 μL of transfection reagent (Pure-fection, System Biosciences) and added to 1.8 mL of complete newt media. Each well of the 6-well plate was incubated in 2 mL of the reagent media for 24 h and then rinsed in newt PBS and complete newt media was added. Approximately half of the A1 cells expressed GFP at 48 h post treatment. After 24 h in complete newt media, the A1 cells were serum-starved as described above. GFP-tagged A1EVs were generated and isolated as described above. For the A1EV tracking study, 5 × 10⁸/mL GFP-labelled A1EVs, were added to SCG neurons in culture for the time described. Treated neurons were fixed in 4% PFA and stained against mouse anti-rat alpha acetylated tubulin (1:300, Sigma), rabbit anti-rat RAB7 (1:100, Abcam) and chicken anti-GFP (1:200, Abcam) primary antibodies and anti-mouse Alexa fluor 647, anti-rabbit Alexa fluor 555, and anti-chicken Alexa fluor 488 secondary antibodies respectively (1:400, Thermo). Images were taken using the Leica TCS SP5 confocal microscope.

To evaluate NK-exosomes internalization by target cells, exosomes were stained with PKH-67 dye (Merck, Darmstadt, Germany) according to [46 (link)] and then incubated with NALM-18 for 30 min, 1 h, 8 h, 14 h, and 24 h. After indicated times, cells were fixed in 4% paraformaldehyde (Sigma Aldrich, Saint Louis, MO, USA) for 5 min and permeabilized with ice cold methanol for 6 minutes. Following adhesion of cells onto poly-L-lysine coated slides, samples were incubated with anti-CD19 antibody (Dako, Carpinteria, CA, USA) for 2 h at RT. After two washes with PBS, slides were incubated with anti-mouse Alexa Fluor 647 (Thermo Fisher, Waltham, MA, USA) and covered with Vectashield antifade mounting medium (Vector Laboratories Burlingame, CA, USA) containing 1.5 µg/mL of DAPI.
Confocal imaging acquisition was performed on Olympus Fluoview FV1000 confocal microscope equipped with FV10-ASW version 4.1a software, Multi Ar (458–488 and 515 nm), 2× He/Ne (543 and 633 nm), and 405-nm diode lasers, using a 60× (1.42 NA oil) objective. Optical single sections were acquired with a scanning mode format of 1024 × 1024 pixels, sampling speed of 12 µs/pixel (pixel size of 0.1 µm), with an electronic zoom at 2. Fluorochromes unmixing was performed by acquisition of automated-sequential collection of multi-channel images, in order to reduce spectral crosstalk between channels.

After oxygen adaptation or DENV2 infection, cells were dissociated with Accutase (STEMCELL Technologies, 07920), washed with PBS, and fixed with 3% paraformaldehyde at 4°C for 30 minutes. Mouse anti-LDLR (1:300, R&D Systems) was then added for 30 minutes at 4°C. After further washing with PBS, anti–mouse Alexa Fluor 647 (Invitrogen, Thermo Fisher Scientific; 1:400) was added and incubate at 4°C for 30 minutes before analysis with the BD LSRFortessa Flow Cytometer.

WI-38 cells or lung microsections (5 μm) were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and blocked in 5% bovine serum albumin for 60 min. The cells or lung microsections were incubated with primary antibody at 4 ℃ overnight, and then incubated with secondary antibody for 1 h at room temperature after three washes with TBST. Nuclei were stained with DAPI for 3 min. Finally, the cells or lung microsections were observed under a laser confocal microscope (Leica TCS SP8; Leica, Wetzlar, Germany). Images from each sections were measured in a blinded manner and quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The following primary antibodies and dilutions were used for immunofluorescence microscopy experiments: α-SMA (1:50, ab7817; Abcam), desmin (1:50, ab15200; Abcam), Ki67 (1:100, 556003; BD Biosciences). The following secondary antibodies were used: anti-mouse-Alexa Fluor 647 (1:1000, Invitrogen), anti-rabbit-Alexa Fluor 488(1:1000, Invitrogen).