Mouse inflammation cytometric bead array cba

Colonic explants were cut longitudinally, rinsed in PBS, and strips of ≈1 cm² tissue and ex vivo biopsies taken from liver (approximately 1 cm³), kidney (one half), and MLN (3 lymph nodes) were placed into 24 flat-bottom-well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 500 μL serum-free RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) as well as penicillin (100 U/mL) plus streptomycin (100 µg/mL; Biochrom, Berlin, Germany). After incubation for 18 h at 37 °C, respective culture supernatants as well as serum samples were tested for TNF-α and IFN-γ by the Mouse Inflammation Cytometric Bead Array (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany).

Ex vivo colonic biopsies of approximately 1 cm² from the colon were longitudinally cut and washed in PBS. Colon tissue and samples of MLN (3 nodes in total) were cultured for 18 h at 37^°C in 24-flat-bottom well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) containing 500 μL serum-free RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), plus penicillin (100 U/mL; Biochrom, Berlin, Germany) and streptomycin (100 μg/mL; Biochrom, Berlin, Germany). Following the manufacturers’ protocol for the Mouse Inflammation Cytometric Bead Array (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany), respective culture supernatant samples were tested for interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1).

Concentrations of cytokines in vaginal lavages were determined using Mouse Inflammation Cytometric Bead Array (CBA) (BD Biosciences) according to the manufacturer's protocol. The results were presented as the means of assays performed in triplicates. Data were analyzed by using FCAP Array 1.0.1 and BD Cytometric Bead Array 1.4 software.

Colonic ex vivo biopsies were cut longitudinally, washed in PBS, and strips of approximately 1 cm² colonic tissue were placed in 24-flat-bottom well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) containing 500 µL serum-free RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL; Biochrom, Berlin, Germany). After incubation for 18 h at 37 °C, colonic culture supernatants as well as serum samples were tested for TNF-α by the Mouse Inflammation Cytometric Bead Array (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany).

Longitudinally cut and washed (in PBS) colonic and ileal ex vivo biopsies (strips of approximately 1 cm²), as well as ex vivo biopsies derived from the MLN (3 nodes), were cultured for 18 h at 37 °C in 24-flat-bottom well culture plates (Thermo Fisher Scientific, Waltham, MA, USA) containing 500 μL serum-free RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), plus penicillin (100 U/mL; Biochrom, Berlin, Germany) and streptomycin (100 μg/mL; Biochrom, Berlin, Germany). Applying the Mouse Inflammation Cytometric Bead Array (CBA; BD Biosciences, Heidelberg, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany), respective culture supernatant samples were tested for interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) according to the manufacturer’s instructions.

Colonic ex vivo biopsies were cut longitudinally, washed in PBS, and strips of approximately 1 cm² tissue as well as ex vivo biopsies derived from MLN (3 lymph nodes), liver (approximately 1 cm³), one kidney (cut longitudinally), and one lung were placed in 24-flat-bottom well culture plates (Nunc, Germany) containing 500 μL serum-free RPMI 1640 medium (Gibco, life technologies, UK) supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL; PAA Laboratories, Germany). After 18 h at 37 °C, culture supernatants were tested for tumor necrosis factor- (TNF-) α, interleukin (IL)-6, interferon (IFN)-γ, and monocyte chemoattractant protein (MCP)-1 by the Mouse Inflammation Cytometric Bead Array (CBA; BD Biosciences, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide was measured by the Griess reaction as reported previously [33 (link)]. Systemic pro-inflammatory mediator concentrations were assessed in serum samples.