Did dye

HUVECs were grown to
90% confluence in 48-well tissue culture-treated plates. LRNVs were
labeled with DiI or DiD dye (Thermo Fisher Scientific). Dyes were
added to LRNVs at a final concentration of 1.67 μM and incubated
at 37 °C for 10 min. Excess DiI was removed using Nanosep centrifugal
ultrafiltration devices with Omega membranes (100 kDa MWCO, Pall OD100C34).
To control for excess DiI micelles, the same procedure was conducted
with the DiI dye alone with the LRNV sample volume replaced with 1×
PBS instead. 5 × 10⁸ LRNV particles were added to
each well and incubated for 4, 6, or 24 h. At the end of the timepoint,
media was aspirated and replaced with fresh media supplemented with
20 μg/mL Hoechst 33342 (Thermo Fisher Scientific #62249) and
imaged using a Carl Zeiss Axio Observer D1 inverted microscope. Particle
uptake was assessed semiquantitatively using ImageJ. Before analysis,
intensities of the control-DiI group were subtracted from the LRNV
group at each timepoint to account for background. For representative
images, cells were plated in eight-well chamber slides (Labtek), fixed
with formalin for 20 min at room temperature (RT), and stained with
DAPI before being mounted and imaged using a Nikon C2 scanning laser
microscope.

Poly (ethylene glycol) diacrylate (PEGDA), hydroxyapatite (HAp), LiBr, APS, TEMED, FITC, and DAPI were purchased from Sigma-Aldrich (USA). Phalloidin, DiD dye, and live/dead kit were purchased from ThermoFisher Scientific. All other chemicals were purchased from Sigma-Aldrich unless otherwise indicated. RUNX2, OCN, OPG, and BSP antibodies were purchased from Santa Cruz (USA). The raw silks were obtained from Simatech Co. (JiangSu, China).

CD11C⁺MHC-II⁺ lung DCs were sorted by flow cytometry. 5 µg/mL DiD dye (Thermo Fisher Scientific) was added to 1 × 10⁶/mL DCs suspended in serum-free RPMI 1640 medium. The DCs were incubated for 20 min at 37 °C and washed twice with RPMI 1640 medium containing 10% FBS. To investigate the trogocytosis process directly, we cocultured 5 × 10⁵ GFP⁺ OVA DNTs and DiD-labeled DCs for 24 h at a ratio of 1:1 in a 24-well plate. For confocal microscopy, the antibodies used were rabbit anti-mouse Lag3 antibody (Abcam) and mouse-purified anti-mouse I-A^b (Biolegend), and DAPI was used as a nuclear stain (Molecular Probes). Donkey anti-rabbit IgG Alexa Fluor 546 and donkey anti-mouse IgG Alexa Fluor 647 (Thermo Fisher Scientific) were used to detect the primary Abs. Confocal analysis was conducted using a confocal laser scanning microscope (FLUOVIEW FV1000, Olympus). The image data were acquired by FV10-ASW 4.2 microscopy software.

hTERT⁺-AM cells were stained with DiD dye (Thermo Fisher, Cat#V22887), according to the manufacturer’s directions. The DiD dye was lost as cells divided, which allowed highly proliferating cells to be identified and distinguished from slowly dividing cells.^{54 (link)–56 (link)} Briefly, 1 mL serum-free DMEM containing 1×10⁶ cells was incubated with DiD dye (1:200) at 37 °C for 10 minutes. After washing three times, the cells were plated in the sterile cell culture dishes and passaged every 2 days. After 10 days, the DiD^high AM cells and DiD^low AM cells were sorted by a FACSVantage instrument (BD FACSAriaFusion) and used for the functional experiment.

Cells cultured for 1 day were fixed with a 3% glutaraldehyde solution. For confocal microscopy, the cells were labeled with DiD dye (D307; Molecular Probes, Thermo Fisher Scientific) and visualized at a 633 nm wavelength using a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany). For scanning electron microscopy, the fixed cells were washed 3 times with deionized water, followed by dehydration with an ethanol gradient (50%, 70%, 90% and 99%) and hexamethyldisilazane treatment (Sigma, St. Louis, MO, USA). The cells were visualized at an acceleration voltage of 5–10 keV in secondary electrons (SE) mode at various magnifications using a Hitachi SU8000 microscope (Hitachi Ltd., Tokyo, Japan).