Tc dish 150

Plasmid combinations of the corresponding VL and VH were cotransfected in HEK293 cells as described elsewhere (53 ). Briefly, cells were seeded in 15% Dulbecco’s modified Eagle’s medium plus GlutaMAX (Gibco) in a TC dish 150 (Sarstedt). The next day, 1 h prior to the transfection, the medium was replaced with Opti-MEM reduced-serum medium supplemented with GlutaMAX (Gibco), and then 120 μg of polyethylenimine MW25000 (Polysciences) was added with 30 μg of the plasmids using the VH and VL vectors at a 2:3 ratio. After 24 h, the medium was changed for RPMI supplemented with 2% ultralow IgG FBS (Gibco). Supernatants from the transfected HEK293 cells were harvested after 4 days, quantified (see the supplemental material), and examined by ELISA and OPA.

For TBARS assay, thiobarbituric acid (TBA), trichloroacetic acid, MDA were purchased from Merck. U373MG cells were seeded into TC Dish 150 (Sarstedt) at a density of 1×10⁵ cells/ml and incubated until confluence. After CAP treatment, cells were further incubated for 24 h, and collected by trypsinisation, centrifuged (100 g for 5 min), and homogenized by sonication. 100 μl homogenate was mixed with 200 μl ice cold 10% trichloroacetic acid and incubated on ice for 15 min to precipitate protein, then centrifuged (2200 g for 15 min at 4 °C). 200 μl of each supernatant was then mixed with 200 μl 0.67% (w/v) TBA and incubate at 100 °C for 10 min. After cooling, samples were measured at 532 nm for MDA.
Lipid peroxidation sensor, C11-BODIPY (581/591) (Thermo Fisher Scientific) was used for in-situ detection and localization of the lipid peroxidation induced by CAP treatment. Cells were incubated in fresh culture medium containing 5 μM of the probe at 37 °C for 30 min in advance. Then the cells were washed with PBS twice and culture medium once. After CAP treatment, cells were further incubated with fresh medium for 30 min at 37 °C, and observed using flow cytometry and confocal microscope as described later.