Cellmask deep red plasma membrane stain

EV internalization by cardiomyocytes was visualized by time-lapse confocal microscopy. hCMs were cultured as described above and seeded onto circular 18 mm coverslips, which are coated with micromolded PDMS. After 5 days in culture, the plasma membrane and nuclei were labeled using CellMask Deep Red Plasma Membrane Stain (Life Technologies) and Hoescht (Life Technologies), respectively, according to the products’ recommended manuals. Following staining, the coverslip was transferred to an imaging chamber filled with 1 mL Live Cell Imaging Buffer (Life Technologies). The microscope (Olympus IX83) was set to perform confocal imaging at several positions along the coverslip, over a period of 3 h, using a 60× objective (Olympus). Immediately after the first time point at which images were acquired, 10⁹ DiO-labeled EEVs were added to the well, and the imaging continued. The acquired images were processed using ImageJ 2.0.0 and Imaris.

Cells were grown in 2-well glass chamber slides at sub-confluent levels and incubated with 5nM GNPs. Cells were stained with CellMask Deep Red Plasma membrane Stain (Life Technologies, C10046) and DAPI (Vector, H-1200). Images were taken with the Zeiss Confocal Laser-scanning Microscope (LSM 510) using a 63× oil immersion objective with a 1.4 NA. The filter sets and laser lines used were: CellMask Deep Red Plasma membrane Stain: excitation 633 nm, BP 650–710; fluorescein: excitation 470 nm BP 500–610; DAPI: excitation 800 nm, BP390-465.

Secretion of mucus by the coculture and its interaction with bacteria
distribution was observed at a concentration of 10³ CFU
mL⁻¹ using laser confocal microscopy (LSM 880, Carl Zeiss
Microscopy, LLC) equipped with an oil immersion objective (PL APO 63x/1.40 oil
DIC, Carl Zeiss Microscopy, LLC). In vitro cocultures were seeded onto sterile
microscope glass coverslips coated with 8 μg cm⁻² of
collagen Type I in 6 well plates at a density of 50 000 cells
cm⁻². After 2 weeks of culture and exposure to
TiO₂ and/or bacteria, cells were fixed with 4% PFA for 30 min at
room temperature and rinsed with PBS. The cell membrane, nucleus and mucus layer
were stained using fluorochromes Cellmask Deep Red plasma membrane stain
(C10046, Life Technologies, USA), Hoechst 33342, and wheat germ agglutinin (WGA,
Alexa Fluor 488 conjugate, Invitrogen) diluted in 1× PBS for 15 min in
the dark at concentrations of 1/1000, 1/500, and 1/200, respectively. Samples
were then rinsed, airdried and coverslips were removed from 6 well plates to be
mounted on a glass slide with ProLong Gold (Thermofisher Scientific, USA) and
dried overnight in the dark. Z-stack images were taken the following day and
images were processed using Zen and ImageJ software (Cornell, Ithaca, USA).

Chitosan (90-150 kDa) was purchased from Sigma-Aldrich (MO, USA). PLGA (acid end cap, 4 kDa, LA:GA=50:50, PLGA₄) was purchased from Durect Corp (AL, USA). PLGA (118 kDa, LA:GA= 65:35, PLGA₁₁₈) was purchased from Lakeshore Biomaterials (AL, USA). PLGA (150 kDa, LA:GA=85:15, PLGA₁₅₀) and fluorescein-conjugated PLGA (7 kDa, LA:GA=50:50, *PLGA) were purchased from Akina Inc. (IN, USA). Paclitaxel (PTX) was a gift from Samyang Genex Corp (Seoul, Korea). LysoTracker Red DND-99, CellMask Deep Red plasma membrane stain, and Hoechst 33342 were purchased form Life Technologies (CA, USA). Methoxy PEG amine, HCl salt (5 kDa, mPEG-NH₂) was purchased from JenKem Technology USA (TX, USA). Dopamine hydrochloride was purchased from Alfa Aesar (MA, USA). Coomassie Brillant blue G-250 protein stain and sodium dodecyl sulfate-acrylamide gel electrophoresis (SDS-PAGE) molecular weight standards were purchased from Bio-Rad (CA, USA).