FIGURE 8:

Cytoplasmic ROS is detected in MH-S cells exposed to silica but not latex particles. MH-S cells were plated in an eight-well chambered slide and labeled with 10 μM CM-H₂DCFDA, and four fields per well were imaged 5 min apart after exposure to either opsonized silica particles or latex particles (A–C). (A) A small increase in fluorescence is observed over time in cells 1 and 2 upon particle addition, which remains constant up to 250 min for cell 1 and 600 min for cell 2 (quantified in C). A further increase in fluorescence is observed after cell blebbing during secondary necrosis (cell swelling) at 550 min for cell 1 and 625 min for cell 2. (B) Comparison of the change in CM-H₂DCFDA fluorescence in control cells and upon exposure to latex or silica particles showing that only silica results in the generation of measurable cytoplasmic ROS. Data represent the mean ± SEM of at least 10 individual cells for each treatment type. (C) Quantification for cells 1 and 2 in A showing a small increase in fluorescence after particle addition as opposed to the huge increase in ROS generation during cell death. (D–F) To detect specifically H₂O₂ in cells, MH-S cells expressing roGFP2-Orp1 were exposed to either opsonized silica or latex particles and imaged at 5-min intervals. (D) A small increase in roGFP2 ratio is observed in first hour; however, this cannot be represented in the ratiometric images. After cell blebbing, a huge change in roGFP2 ratio is observed at 330 min. (E) Comparison of the change in roGFP2-Orp1 ratio in control cells and upon exposure to latex or silica particles showing that only silica results in the generation of measurable cytoplasmic H₂O₂. Data represent mean ± SEM of at least 10 individual cells for each treatment type. (F) Quantification for the cell in D is represented by cell 1, and other cells are represented as 2–6. All of these cells show a huge increase in ratio at different time points, which relates to cell death. (G) To detect mitochondrial H₂O₂, MH-S cells expressing mito-roGFP2-Orp1 were exposed to opsonized silica particles. In pseudocolored ratiometric images, mitochondria appear red to yellow, indicative of basal H₂O₂, whereas upon secondary necrosis, there is an increase in H₂O₂ from 420 min onward; mitochondria appear blue to pink. (H) Quantification of cell represented in G, cell 1. Other cells show a similar trend, with a huge increase in ratio observed during secondary necrosis after 350 min.