Il 1beta elisa kit

The wild-type human monocytic THP-1 cell line (designated Null-1) and gasdermin D knockout THP-1 cell line (THP1-KO-GSDMD) were both obtained from InvivoGen (Toulouse, France). The cells were cultured in RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, glutamine (2 mM), Normocin TM (100 mg/mL) and Zeocin TM (100 mg/mL) selection antibiotics mixture (InvivoGen, France). THP-Null 1 and THP1-KO-GSDMD cells were differentiated into macrophagelike cells for 72 h using 50 nM phorbol 12-myristate 13-acetate (PMA; Sigma, Sweden). After differentiation, cells were exposed to MWCNTs (NM401) at 25 mg/mL for 24 h. After exposure, the cell supernatant was collected and IL-1b release was quantified using the IL1beta ELISA kit (Invitrogen, Sweden). To further validate the role of GSDMD in wild-type THP1-cells, cells were differentiated for 72 h with 50 nM PMA and then preincubated with 30 mM disulfiram (Sigma, Sweden) for 1 h and exposed to MWCNTs for 24 h. In parallel, nigericin (50 mM, Sigma-Aldrich, Sweden) was applied as a positive control to induce inflammasome mediated IL-1b release in LPS-primed (0.1 mg/mL) cells.

Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in the perfusate were measured 1 h after perfusion using commercially available enzyme-linked immunosorbent assay kits (TNF alpha ELISA Kit, Rat; IL-1 beta ELISA Kit, Rat; and IL-6 Rat ELISA Kit; Invitrogen Corporation, Camarillo, CA, USA).