Complete rpmi 1640 culture medium

Magnetic bead separation (130-049-201, Miltenyi Biotec, Germany) were utilized to isolate mice spleen CD4⁺ T cells from untreated healthy mice using the protocol reported in our previous studies 14 (link), 25 (link). BECs were pretreated with 1 nM of E2, 20 nM of DHT and 20:1 nM of DHT:E2 in the asthma+E2, asthma+DHT, asthma+DHT+E2 groups for 24h, respectively. Then BECs were cocultured with CD4+ T cells at a ratio of 10: 1 (TCs: BECs) 13 for 24h with complete RPMI 1640 culture medium (Gibco, Australia) supplemented with 10% FBS, 1% penicillin and streptomycin, soluble anti-CD28 (1.0 μg/ml, eBioscience), soluble anti-CD3 (0.5 μg/ml, eBioscience), and IL-2 (20 ng/ml, eBioscience) in transwell plate (Corning, USA). After 24h, cells were collected and then detected by flow cytometry.

The HDACi (LBH589, SAHA and FK228) were purchased from Selleck Chemicals, Munich, Germany. The MAPK inhibitors, GSK1120212 and ARRY-162, were obtained from Selleck Chemicals and AbMole BioScience, HongKong, China, respectively. FAK inhibitor TAE226 was from Selleck Chemicals and PF573228 was from Tocris Bioscience, Bristol, UK. Phosphatidylinositol 3-kinases (PI3K) inhibitors GDC-0941 and BKM120 were obtained from Selleck Chemicals. All compounds were dissolved in dimethyl sulfoxide and stock drug solutions were diluted in complete RPMI-1640 culture medium (Life Technologies, Mulgrave, VIC, Australia) to various concentrations for experimentation.

Buffy coats were sourced from the Australian Red Cross Lifeblood as approved by the University of Melbourne Human Research Ethics Committee (2021-20542) and all methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardian(s). Human monocytes were isolated by negative selection using the RosetteSep Human Monocyte Enrichment cocktail (Stem Cell Technologies, Vancouver, BC, Canada), as before^{48 (link)}. Negatively selected monocytes were further enriched by performing two rounds of ‘platelet wash’ with centrifugation at 120×g for 10 min at room temperature with no brake. The purity of the enriched monocytes was found to be consistently above 85%, as ascertained by flow cytometry with anti-human CD14-PerCP-Cy5.5 antibodies and IgG1k-PecCP-Cy5.5 as isotype control (Thermo Fischer Scientific, Waltham, MA). Isolated monocytes were resuspended in 10 mL of complete RPMI 1640 culture medium, containing 10% heat inactivated fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1% GlutaMax-1 (Life Technologies, Carlsbad, CA). Cell count and viability were assessed using Trypan blue staining and counted via Countess II Automated Cell Counter (Thermo Fischer Scientific). Monocytes were cultured with P. falciparum-iRBC or malaria pigment Hz at 37 °C in a 5% CO₂ humidified environment.

PBMCs, isolated from the healthy subjects, were cultivated in RPMI 1640 complete culture medium (Gibco Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Gibco Life Technologies), 1% penicillin-streptomycin (Gibco Life Technologies) and 5.6 mM glucose at 37.0°C in a humidified atmosphere (5% CO₂, 95% air). Following overnight culture in six-well plates, the PBMCs were incubated with and without 250 μM PA (Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The cells were harvested for RNA and protein expression analysis, and the supernatant was collected for TNF-α and IL-6 measurement by ELISA.

LN229 and T98G cell lines were purchased from American Type Culture Collection (ATCC) and maintained in RPMI 1640 complete culture medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). TPH-1 overexpressing LN229 and T98G cells were generated by Cyagen (China) and the expressions were determined by western blotting. Temozolomide (TMZ) and serotonin were purchased from Sigma (USA). The NF-κB inhibitor, QNZ (EVP4593), was obtained from Selleck (USA). TPH-1 inhibitor, LX-1031, was obtained from Abcam (UK).