E el h6156

Serum levels of estrogen were measured by ELISA using mouse estrogen kits (EM1501, FineTest, Hubei, China). Serum levels of the cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, were measured by ELISA using mouse-specific kits (R&D Systems, MTA00B, MLB00C, M6000B).
HUVEC were treated with different compounds at a density of 5 × 10⁵cells/well for 24h (seeded in 12-well plates). Then cell culture supernatants were collected and centrifuged at 1000 × g at 4°C for 20 min, and supernatants were then used to determine the levels of IL-6, IL-1β and TNF-a, in accordance with the manufacturer’s instructions (Elabscience, Wuhan, China, E-EL-H6156, E-EL-H0149c, E-EL-H5548c). All samples were assayed in triplicate.

To detect secretion of IL-1β, IL-6, IL-8, and TNF-α in the supernatant, we plated cells in 6-well plates and subjected them to various treatments. After centrifugation at 1000 rpm for 10 min, the supernatant was collected. The levels of secreted IL-1β (E-EL-H0149c), IL-6 (E-EL-H6156), IL-8 (E-EL-H6008), and TNF-α (E-EL-H0109c) (all from Elabscience, China) were determined with ELISA kits according to the manufacturer’s instructions.

Concentrations of human TGF-β (Elabscience, Wuhan, China, Cat#E-EL-0162c), TNF-α (Elabscience, Cat#E-EL-H0109c) and IL-6 (Elabscience, Cat#E-EL-H6156) secreted from the irradiated cells were measured with ELISA kits, in accordance with the instructions of the manufacturer. Briefly, cell culture media were collected and centrifuged at 1000 g (20 min, 4 °C) to remove cell debris. The supernatants were collected for ELISA assays.

The HBE cell culture medium was collected and centrifuged for 5 min at 3,000 rpm. The supernatant was centrifuged and assayed according to the manufacturer’s instructions for the glutathione (GSH) kits (A006-2-1, Jiancheng Bioengineering Institute, Nanjing, China). The levels of IL-6, TNF-α, and prostaglandin E2 (PGE2) were measured according to the manufacturer’s kit instructions (E-EL-H6156, H0109c,0034c, Elabscience, Wuhan China), and the cell assays were repeated three times.

Sex- and age-matched groups of 32 patients with BAVMs and 32 controls were selected to measure WBC MTA1 protein expression. Cell supernatants were used to detect the expressions of IL-6 and TNF-α. The ELISA kits were used according to the manufacturer′s instructions to detect MTA1 protein levels (MULTISCIENCES, Hangzhou, China), IL-6 (E-EL-H6156, Elabscience, Wuhan, China), and TNF-α (E-EL-H0109c, Elabscience). A multifunctional microplate reader (Molecular Devices, CA, USA) was used to record the absorbance of each well at 450 nm.

10 ml of venous blood was collected using venipuncture at different time points, including resting, immediately after completion, 45 min post-SIT (early response), and 24 and 48 h after SIT (late response). The blood sample was spun at 4ºC for 15 min at 1000 × g within 30 min of collection, and the separated plasma was stored at −80ºC and was analyzed subsequently. CK concentrations (MyBioSource, MBS269244; San Diego, California, USA), Interleukin 6 and 10 (IL-6 and IL-10), and tumor necrosis factor-alpha (TNFα) (Elabscience, E-EL-H6156, 6154, and 0109, respectively; Houston, Texas, USA) were analyzed using ELISA assays per manufacturer instructions. The coefficients for variation were <10%, and the sensitivities were 0.06 ng/mL, 0.93 pg/mL, 0.94 pg/mL, and 4.69 pg/mL, respectively.