Ab100712

Liproxstatin-1 (S7699, Selleck, TX, USA), a ferroptosis inhibitor, was administered i.p. at a concentration of 10 mg/kg 1 h before ischemia induction, in accordance with previous study protocols [9 (link)]. Mice were killed at 30 min of reperfusion and serum was collected from the abdominal aorta. In addition, Liproxstatin-1 dissolved to a final concentration of 200 nM was used to treat Caco-2 cells in vitro for 12 h before hypoxia induction. Rosiglitazone (ROSI, S2556, Selleck), a classic peroxisome proliferator-activated receptor-γ agonist that has been used for ACSL4 inhibition, was administered intravenously at a concentration of 0.4 mg/kg 1 h before ischemia induction, as pretreatment of ROSI allows sufficient time for proper phospholipid remodeling in the membranes [21 (link), 22 (link)]. Mice were killed at 45 min of ischemia or at 30 min of reperfusion. Serum was collected from the abdominal aorta. Kits were used to assay the levels of tumor necrosis factor (TNF)-α (ab208348, Abcam, MA, USA), interleukin (IL)-6 (ab100712, Abcam), and ACSL4 activity (ab241005, Abcam).

Mouse serum levels of IL-1β, IL-6, and IL-8 were determined using the ELISA kit for IL-1β (ab100704, Abcam, Cambridge, UK), IL-6 (ab100712, Abcam), and IL-8 (SEKM-0046, Solarbio, Beijing, China), referring to the kit instructions [20 (link)]. Absorbance was obtained at 450 nm using a microplate reader (800TS, BioTek, Winooski, VT) and analyzed using the Origin 9.5 software.

According to the instructions, ELISA was used to detect the secretion of inflammatory factors in the supernatant of FLSs and mouse serum: IL-6 (ab100712), IL-1β (ab197742), and TNF-α (ab208348) (Abcam).

The levels of TNF-α (ab208348, Abcam Inc., Cambridge, UK), interleukin (IL)-1β (ab100704, Abcam Inc., Cambridge, UK), and IL-6 (ab100712, Abcam Inc., Cambridge, UK) in serum of mice in each group were determined according to the manufacturer’s instructions of the ELISA kit (Linco Research Inc., St Charles, Missouri, USA).

Quantification immunomodulatory cytokines released from splenocytes was determined using commercially available enzyme-linked immunosorbent assay (ELISA). The ELISA kits detecting murine interleukin-3 (IL3, ab113345) and IL6 (ab100712) were obtained from Abcam (Cambridge, MA, USA). The kits for murine interferon-gamma (IFNγ, MIF00) and tumor necrosis factor-alpha (TNFα, MTA00B) were obtained from R&D systems (Minneapolis, MN, USA). The culture medium was clarified by centrifugation at 12,000 rpm for 1 min, and the supernatant was subjected to ELISA assay following the manufacturer’s instruction. Horseradish peroxidase-mediated 3,3',5,5'-tetramethylbenzidine color development was quantified at 450 nm using the Emax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The concentrations of the cytokines released in the medium were quantified by comparison with serially diluted standards that were included in the kits.