To measure the extent of particle internalization into cells, three methods were used: FACS analysis, spectrometry microplate reader analysis, and microscopy imaging. Measurements of uptake after 24 hours incubation with fluorescently labeled polystyrene particles were performed. Control experiments were done with 5-min incubation, ensuring low particle-cell affinity that does not depend on particle size (fig. S5).

For FACS analysis, cells were seeded in six-well plates for 4 days after overnight starvation in low (0.5%, v/v) serum media. Fluorescently labeled polystyrene particles were added and diluted either to final concentrations of 0.0007 to 0.003% (w/v) (Fig. 2A) or to fixed concentrations of 5% (v/v) otherwise, for O/N incubation in fresh full medium. Controls of cells without particles in the same conditions were done as well. The minimal number of cells in a sample was 10,000. Normalized uptake values were calculated using eq. S1. After the incubation with particles, the cells were washed with cold phosphate-buffered saline (PBS), detached using trypsin, washed again, and filtered through a 40- to 50-μm nylon mesh using a 50-ml conical tube to remove tissue debris mesh. Cells were then centrifuged and suspended in a FACS buffer containing 1% bovine serum albumin in PBS and 0.05% sodium azide. Analyses were performed using a Beckman Coulter CytoFLEX (USA) flow cytometer and analyzed using CytExpert software.

Further quantification of particle uptake by cells was done by fluorescence detection using a microplate reader (Synergy, HT, BioTek, USA). The tested cells were seeded in a final concentration of 20,000 cells per well in 96-well clear-bottom plates (Corning, Sigma-Aldrich) and cultured until cells reach 90% confluency. Polystyrene beads were added from 1% (w/v) stock to a final concentration of 5% (v/v). Normalized uptake values were calculated using eq. S1. Cells were incubated with beads for 24 hours, washed thoroughly with PBS, and read with the microplate reader in excitation: 530/25 and emission: 590/20. Cells without beads were used to obtain a baseline signal.

Cell imaging was used as a direct detection of particle uptake that we used, both with an inverted fluorescent microscope (model IX73, Olympus Corporation, Japan) and by confocal microscopy (Nikon’s A1 MP multiphoton confocal microscope equipped with a 639-nm diode). For fluorescence microscopy, cells were seeded in 24-well plates. After O/N starvation (serum-free medium), fluorescently labeled beads, in the size of 0.05 to 2.4 μm, were added for an O/N incubation. The cells were then washed repeatedly, fixed using 4% paraformaldehyde (PFA), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Wells were then observed and photographed using fluorescent microscopy (Olympus IX73).

To further validate the extent of particle uptake, and differentiate between internalization and cell adhesion, a confocal microscope with optical sectioning was used. Cells were seeded in ibidi eight-well microplates for 3 days, during which cells were starved overnight and later incubated O/N with fluorescent-labeled beads ranging from 0.05 to 2.4 μm in fresh full medium. Cells were then washed thoroughly, fixated with 4% PFA, and stained with DAPI for nuclei staining with or without counterstaining of Alexa Fluor 488 phalloidin for actin staining. The wells were imaged and photographed using a ZEISS (Germany) confocal microscope. Z-stacks (~1 μm per slice) were performed for ortho and 3D analysis using ZEISS Zen software.

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