Fusion sl2 3500 wl

Total protein lysates from ischemic colonic anastomotic tissue were prepared using T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) and Tissue Lyser II (Qiagen). Lysates were then analyzed with a Mouse XL Cytokine Array (ARY028, R&D Systems, Bio-Techne). Chemiluminescence was detected with Fusion SL2-3500.WL (Vilber Lourmat). Densitometric analysis of dots of each cytokine was performed with ImageJ. Background signal was subtracted, and expression of each cytokine was normalized to its own positive control.
In addition, IL-6 protein levels were determined in whole tissue lysates from septic colonic anastomoses using a commercially available ELISA (M6000B, R&D Systems, Bio-Techne). In CM from LPS-polarized proinflammatory (M1) BMDMs, IL-6 protein and CXCL-1 were also analyzed by ELISA (IL-6: M6000B, R&D Systems, Bio-Techne; CXCL-1: MKC00B, R&D Systems, Bio-Techne). Absorbance was measured at 450 nm. Further details on culture conditions are outlined below. All experiments were performed in triplicate.

Peritoneal fluid was analysed with a Mouse XL Cytokine Array (R&D Systems) according to the manufacturer’s instructions. Chemiluminescence was detected with Fusion SL2-3500.WL (Vilber Lourmat, Suebia, Germany). Densiometric analysis of dots representing each cytokine was performed with ImageJ. Background signal was subtracted, and expression of each cytokine was normalized to its own positive control. Levels of IL-6 (R&D Systems), PAI-1 (Thermo Fisher Scientific), active tPA (LifeSpan BioScience, Seattle, USA) and were quantified by ELISA. Absorbance was measured at 450 and 540 nm using a 96-well plate reader (Tecan, Crailsheim, Germany).