Celltrace calcein green

30,000 cells/well were seeded in eight-chamber slides. Cells were washed with PBS at the harvesting time point and fixed with 4%parafornaldehyde for 25 min. Cells were then washed with PBS and permeabilized with 0.2% Triton X-100 for 5–10 mins. Cells were then washed with PBS and incubated overnight 1:100 with the indicated primary antibody cytochrome C (#sc-13560; Santa Cruz; RRID:AB_627383), Tom-20 (#42406, Cell Signaling Technology; RRID:AB_2687663), cells were washed with PBS and incubated with secondary antibody 1:200 goat anti-mouse Alexa Fluor 488 (#A-11008, Thermo Fisher Scientific; RID:AB_143165) and Cy3 AffiniPure Donkey anti-rabbit (#711-165-152, Jackson Immuno Research) for 1 hr followed by PBS washed, 1:400 DAPI staining, washed with PBS and imaged. Organoid viability imaging was determined by CellTrace Calcein Green (#C34852, Thermo Fisher Scientific), Ethidium Homodimer-1 (#E1169, Thermo Fisher Scientific), Ki-67 (#9449; Cell Signaling Technology; RRID:AB_2797703) incubated at 37°C for 1 hr then washed with PBS and imaged. Imaging was done using a Leica Confocal Microscope. Experiments were performed at least twice and more than three technical replicates were obtained, a representation of one is shown.

PBS, CFS, or NLS (20 μL) containing 0.5 mg/mL of 10-kDa
dextran-tetramethylrhodamine (Life Technologies) was added to the apical surface
of NHBE. Cells were labeled with CellTrace Calcein Green, AM (Thermo Fisher; 5
µg/mL in media). ASL height was stabilized by 120 min. Vasoactive
intestinal peptide (100 nM) (VIP; Life Technologies) was added basolaterally to
induce CFTR-mediated secretion. Perfluorocarbon (3 M Fluorinert FC-770) was
added apically to prevent ASL evaporation. Images were obtained immediately
before and 60 min after basolateral VIP addition in XZ-scanning mode by using a
Leica SP8 confocal microscope with a ×63/1.3 numerical aperture (NA)
glycerol immersion lens. Ten ASL images per culture were acquired by using an
automatic stage with the “Mark-and-Find” function as described
(16 (link)).

EVs isolated in the P40 fraction were stained for 30 min at room temperature with 0.5 μm CellTrace Calcein Green (ex488/em521), AM (Thermo Fisher Scientific), and/or 0.5 μm Potomac Gold (ex561/em594) in 20 mm Tris-HCL, pH 6.0. Potomac Gold was acquired from Luke Lavis (HHMI Janelia). The stained samples were purified using an iodixanol density gradient as described above. The stained EV samples were mounted on 0.1% poly-l-lysine–coated Cellviz dishes (Cat. # D35-10-1.5N), and the background fluorescence of Potomac Gold was quenched using 50 nm trypan blue in HPLC water. Samples were imaged using Borealis total internal reflection fluorescence (BTIRF) or spinning disk confocal microscopy on an Andor Dragonfly 600 microscope (Oxford Instruments) with an Andor Zyla 4.2 PLUS sCMOS camera and a Leica Plan Apo 63× oil immersion TIRF objective (numerical aperture, 1.47). A 2,000 mW, 488 nm excitation laser at 3% and a 521/38 nm bandpass (BP) filter with an exposure time of 500 ms and an averaging of 4 was used for Calcein AM. For Potomac Gold, a 1,000 mW, 561 nm laser at 5% and 594/43 nm BP filter with an exposure time of 500 ms and an averaging of 4 was used.