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...
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 (...
To understand how immune checkpoint perturbation impacts the colonic immune system, we administered anti-CTLA4/anti-PD-1 combination therapy to wild-type (WT) Balb/C mice. Combination checkpoint inhibition triggered systemic immune activation with induction of splenomegaly, even after 1 week of treatmen...
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 ...
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...
ELISA was performed according to the manufacturer’s instructions. The absorption was measured at 450 nm. The protein concentration was determined by comparing the relative absorbance of the samples with the standards. Each result was repeated three times. Quantitative real-time PCR (RT-qPCR) RT...
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
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...
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