NOS activity was measured by an ultra- sensitive colorimetric assay (Oxford Biomedical Research, Rochester Hills, MI, cat. no. NB 78) as per the manufacturer's instructions. ROS staining. LP cells were stained for surface markers, incubated for 30 min at 37 1C with 10 mM CM-H2DCFDA (...
Total RNA was isolated from highly pure, sorted human ILC subsets using the TRIZOL reagent according to the manufacturer’s instructions (Invitrogen). Final preparation of RNA was considered DNA- and protein-free if the ratio of readings at 260/280 nm was ≥1.7. Isolated mRNA was reverse-tr...
DNA was extracted from stool using the DNeasy Powerlyzer Powersoil Kit (Qiagen) following manufacturer instructions with an additional bead beating step for three minutes at speed 8 in a Bullet Blender Storm (Chembio Ltd, St Albans, UK). DNA was quantified using a Qubit Fluorometer (Thermo...
according to the manufacturer’s instructions. Reverse tran- scription and real-time PCR reactions were then carried out using LightCycler-RNA Master Hybridization Probes (one- step procedure) on a LightcyclerR apparatus (Roche Diagnostics, Brussels, Belgium). Primers, standards, and real-time PCR...
Immunohistochemical staining was performed using the fully automated immunostainer Benchmark XT (Roche, Basel, Switzerland). Staining protocol included deparaffinization and counterstain with hematoxylin and blueing reagent according to the manufacturers´ instructions. The IL-6 antibody required pretreatment ...
and total RNA was isolated according to manufacturer's instructions. For FACS-sorted cell mRNA isolation, RNeasy kit (Qiagen) was used. Complementary DNAs were prepared using SMART MMLV reverse transcriptase (Clontech) and used for PCR with SYBR Green reagents (Qiagen or Quanta Bioscience) on a...
Immune checkpoint inhibitors (CPIs) are a relatively newly licenced cancer treatment, which make a once previously untreatable disease now amenable to a potential cure. Combination regimens of anti-CTLA4 and anti-PD-1 show enhanced efficacy but are prone
Cells were transfected with either specific siRNA or scrambled siRNA using Amaxa Mouse Macrophage Nucleofector Kit from Lonza following manufacturer's instructions. Post-transfection, cells were maintained at 37 °C in the presence of 5% CO2. The efficiency of gene knockdown was verified by qRT-PCR...
Telomere in situ hybridization and SKY analyses were performed using Cy3- labeled telomere PNA (CCCTAA)3 probe (Panagene Inc., Daejeon, Korea) and 20-color mouse SKY paint kit (ASI), respectively, following standard procedures and manufacturer's instructions.39 SKY images were acquired using a ...
ST2hi memory-type Th2 cells are identified as a pathogenic subpopulation in eosinophilic airway inflammation. These ST2hi pathogenic Th2 cells produce large amount of IL-5 upon T cell receptor stimulation, but not in response to IL-33 treatment. By contr