Formaldehyde glutaraldehyde 2

After removing the basolateral media (24 hours upon exposure), the cells were detached using TRYPLE. After 3 min incubation, 0.7 mL of DMEM with FBS was added, and the cells were resuspended. The suspension was transferred to a vial and centrifuged at 1.2 K xg for 5 min. The supernatant was removed, and 0.7 mL fixation buffer (formaldehyde glutaraldehyde 2.5%, Electron Microscopy Sciences, PA, USA) was added, and the suspension was vortexed for 5 min. The suspension was left to rest at room temperature for 15–30 min, followed by storage at 2–8 °C. The fixed samples were cut and prepared for TEM analysis. The TEM samples were imaged with transmission electron microscopy (FEI TECNAI T-12, Thermo Fischer Scientific, MA, USA), and they were evaluated qualitatively for differences in uptake.

24 h after dosing the co-culture with combustion particles, the basolateral media was removed, and the cells were detached after 3 min incubation in TRYP-LE. The cells were resuspended in 0.7 mL of DMEM with FBS. The suspension was transferred into a vial and centrifuged at 1200×g for 5 min. The supernatant was removed, 0.7 mL fixation buffer (formaldehyde glutaraldehyde 2.5%, Electron Microscopy Sciences, PA, USA) was added, and the suspension was vortexed for 5 min for mixing. The suspension was left to rest at room temperature for 15–30 min, followed by storage at 2–8 °C. The fixed samples were fabricated and prepared for TEM analysis. The images were taken using FEI TECNAI T-12 TEM operated at 120 kV (Thermo Fischer Scientific, MA, USA). A qualitative analysis of uptake was made between air controls and the BSC and ASC treated cells.