Alexa fluor 647

Single-cell suspensions were prepared from 4-day cultures of breast reduction samples, and Epithelial Cell Adhesion Molecule positive (EpCAM⁺) cells were immunomagnetically isolated (> 90% purity, Additional file 1: Figure S4C) as described before [1 (link)] and cells were stained with anti-α6 integrin (CD49f), conjugated to alexafluor 647 (clone# G0H3, Biolegend.), anti-CD90, conjugated to phycoerythrin (PE) (clone# 5E10, Biolegend), anti-NOTCH3 (clone# 603532, R&D systems), and anti-FZD7 (clone# 151143, R&D systems) antibodies. A goat anti-rat fluorescein isothiocyanate (FITC) was used to detect FZD7 protein, a goat anti-mouse Alexa Fluor 405 (Invitrogen) was used to detect the NR3 protein, and propidium iodide (PI) exclusion was used to identify live cells. The bulk bipotent progenitors (BP, EpCAM⁺CD49f⁺CD90⁺), BP-subset “a” (EpCAM⁺CD49f⁺CD90⁺ NR3^highFZD7⁺), BP-subset “b” (EpCAM⁺CD49f⁺CD90⁺NR3^−/lowFZD7⁺), BP-subset “ab” (EpCAM⁺CD49f⁺CD90⁺NR3^mediumFZD7⁺), bulk luminal-restricted progenitors (LRPs, EpCAM⁺CD49f⁺CD90⁻), LRP-subset “a” (EpCAM⁺CD49f⁺CD90⁻NR3⁺FZD7⁺), and the LRP-subset “b” (EpCAM⁺CD49f^l+CD90⁻NR3⁻FZD7⁺) were isolated via Fluorescent Activated Cell Sorting (FACS).

The protocol for bead-assisted
flow cytometry for sEVs was adapted from Thery et al.(50 ) Unlabeled or ⁸⁹Zr-labeled
PANC1 sEVs (intact or heat-damaged) at a concentration of 1 ×
10¹⁰ sEVs in 40 μL of PBS were incubated with 10
μL of aldehyde/sulfate latex beads (3.9 μm, 4% w/v; Molecular
Probes) for 15 min at RT. 10 μM BSA was added to the sEV-bead
mixture and incubated for 15 min at RT. 1 mL of PBS was added and
incubated for further 75 min at RT on an orbital rotator. The beads
were pelleted by centrifugation for 5 min at 600g, re-suspended with 1 mL of 100 mM glycine in PBS, and incubated
for 30 min at RT. The beads were washed twice with 2% FBS in PBS (FBS/PBS).
Aliquots of the sEV-bead suspension were incubated with 1 μg
of mouse anti-human CD63 (BioLegend #353013), CD81 (BioLegend #349520),
and CD9 (BioLegend #312102) antibodies in separate tubes or with no
primary antibody (2° only control) for 40 min at 4 °C. The
beads were washed once, re-suspended in FBS/PBS, and incubated with
goat anti-mouse AlexaFluor 647 (0.5 μg/mL; BioLegend #405322)
for 40 min at 4 °C, covered in foil. Finally, the beads were
washed and suspended in 200 μL of FBS/PBS for flow cytometry
analysis on FACS Melody (BD Biosciences), and the data were analyzed
on FlowJo v10. The 2° only population was used for gating control.