Tanon 5200 chemiluminescence image analysis system

HL60 received RSL3 for 6 h to induce ferroptosis. THP-1 cells were pretreated with PMA (40 pmol/10⁶ cells) for 3 days and incubated with ferroptotic cells for 1.5 h at 37 °C. Unengulfed HL60 cells were removed. Then, the THP-1 cells were treated with 50 μM biotin or SAPE-biotin in RPMI 1640 (without FBS) for 4 h, and then harvested and incubated with prewashed streptavidin beads (Thermo Fisher Scientific) overnight at 4 °C on a shaker. The beads were collected and heated to 100 °C for 10 min, and then loaded on SDS-PAGE.
For protein gel staining, gels were incubated with Coomassie Brilliant Blue staining solution for 6 h, and then washing five times with 1% phosphoric acid (H₃PO₄).
For western blotting, proteins were transferred from gel to Immobilon-P PVDF membrane (MilliporeSigma). Proteins expression were detected using TLR2 antibody and visualized using secondary antibody conjugated with HRP and Pierce ECL western blotting substrate (Thermo Fisher Scientific) as the substrate of HRP. The immunoblot was detected by Tanon 5200 Chemiluminescence Image Analysis System (Tanon Science & Technology).

PC12 cells were treated with DAQ probe (DA-yne probe contains FeCl₃ solution) or DMSO for 6 hours, and then were harvested and lysed with RIPA buffer supplemented with protease inhibitor cocktails and phosphatase inhibitor cocktails. A freshly premixed click chemistry reaction cocktail (50 μM TAMRA-N₃ from 25 mM stock solution in DMSO, 100 μM TBTA from 50 mM stock solution in DMSO, 1 mM TCEP from 1 M stock solution in deionized water, and 1 mM CuSO₄ from 1 M stock solution in deionized water. All the stock solutions above were freshly prepared.) was added to equal amounts of protein lysate. The reaction mixture was further incubated at 25°C for 2 hours, and the solution was transferred to a centrifugation tube where prechilled acetone at –20°C was added and the solution was then dissolved in PBS + 1% SDS. Upon incubation with streptavidin beads at 25°C for 4 hours, 2 × 100 μL SDS loading buffer was added and heated at 95°C for 10 minutes. Proteins were separated by SDS-PAGE gel and transferred to Immobilon-P PVDF membrane (Sigma-Aldrich). Protein expression was detected using GPX4 antibody and visualized using secondary antibody conjugated with HRP and Pierce ECL Western blotting substrate (Thermo Fisher Scientific) as the substrate of HRP. The immunoblot was detected by Tanon 5200 Chemiluminescence Image Analysis System (Tanon Science & Technology).