6 well plate

Established biofilms were first grown as follows. 300 μl of log-phase culture inoculation was added to 5 ml BHI media in wells of untreated 6-well plate (Celltreat Scientific Products, 229506), where biofilms were grown for 3 to 7 days by immersing 22 mm × 22 mm plastic coverslips (Carolina Biological Supply Company, 632900) in the solution. To study the effect of MMP1, 500 μl of 1 mg/ml MMP1 was added in each well. For control experiments, 500 μl of BHI media and protein buffer were added instead of MMP1. To image biofilms, the solution was aspirated and coverslips were washed three times with 500 μl of sterile PBS and dried at 37°C for 24 hr before imaging using a Phenom Pro-Scanning Electron microscope.

U-2 OS cells were cultured and plated in a 6-well plate (Celltreat, Pepperell, MA, USA). Once adherent, cells were treated with 5 μg/mL of 0.1 μm PS-MPs or 20 μg/mL of 2 μm PS-MPs for 24 h. After exposure, the cells were washed with sterile PBS (Gibco, Waltham, MA, USA), detached, centrifuged in DMEM (Gibco, Waltham, MA, USA) at 1000× g at 4 °C for 5 min to remove excess PS-MPs, and re-plated on 13 mm coverslips in a 6-well plate. After 24 h, the cells were washed 3 times with PBS, fixed for 10 min with 4% formaldehyde at RT in PBS, permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS at RT for 15 min, stained with Hoescht (1:2000, H1399, Invitrogen, Waltham, MA, USA) and Phalloidin (1:500, PF7501, EPM Scientific, New York, NY, USA) for 5 min at RT, washed 3 times in PBS, and mounted onto glass slides with an aqueous mounting medium. Fluorescence imaging (Leica THUNDER DMi8 3D Fluorescence Imaging System, Leica Biosystems, Wetzlar, Germany and LAS X 3D Analysis Software v. 2018.7.3, Leica Biosystems, Wetzlar, Germany) was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.

RAW264.7 cells were cultured at high and low density (2500 and 400 cells/mm² respectively) overnight in a 6-well plate (CellTreat 229106) in DMEM media supplemented with 10% FBS. Cells at each density were divided into five aliquots of equal cell count (approximately 0.5 x 106) and washed with PBS, stained with a live dead reactive dye (BioLegend L34975), fixed and permeabilized with Cyto-Fast Fix/Perm Kit (BioLegend 426803) following kit instructions, and then blocked with anti-mouse CD16/32 antibody (BioLegend 101319) at 10 ug/mL. For each density, three samples were stained with PE anti-mouse CD287 (TLR7) Antibody (BioLegend 160003) at 5 ug/mL, one with PE Mouse IgG1, κ Isotype Ctrl (FC) Antibody (BioLegend 400113) at 5 ug/mL, and one left unstained, at the steps indicated in the Fix/Perm protocol. The cells were then washed and resuspended in Cell Staining Buffer (BioLegend 420201) and run on an ACEA NovoCyte Flow Cytometer.