A selected list of state-of-the art tools and techniques we use to answer research questions in the lab
IN VIVO TOOLS
Models of colitis (DSS, CD45RBhi adoptive transfer colitis); Models of hepatic fibrosis; Tumor models (e.g. melanoma); Adoptive cell transfer models; Viral infection models; In vivo T cell conversion assays; Experimental autoimmune encephalomyelitis assay (EAE); Bone marrow chimeras; In vivo cytokine neutralization; Measurement of intestinal barrier integrity (FITC-dextran and radioactive labeled Mannitol); Injections and oral gavage; cheek bleeding; Ear-swelling measurements to evaluate systemic immune responses to antigens
IN VITRO SYSTEMS
Hematopoietic stem cell culture systems; In-vitro cell culture of primary human and mouse lymphocytes and variety of cell lines including mouse/human tumor cell lines; co-cultures of T cells with antigen presenting cells and tumor cells combined with various stimulation/treatment protocols; in vitro T helper cell differentiation assays; transfection (electroporation, lipofection) and retroviral transduction of various primary human and mouse immune cells and cell lines; 3H-Thymidine, CFSE, Ki67 and BrdU proliferation assays; human and mouse immune cell isolation (and Percoll gradients) from various organs (spleen, lymph nodes, mLN, intestine, blood, mouse/human intestinal biopsies, bone marrow, brain, spinal cord, and tumors).
Flow cytometry (+20 color CYTEC AURORA, FACSDiva, Flowjo); cell sorting (FACSAria); Mass-spectroscopy (GC-MS); Electron microscopy (SEM)
Anaerobic (anaerobic chamber) and aerobic culture of organs to identify systemic bacterial dissemination in immune organs and identify bacterial species by sequencing of 16Sr RNA gene
Germ free mice, Fecal microbial transfer studies; Test the requirement of the gut microbiota in the development of diseases
Immunohistochemistry (IHC) and Immunocytochemistry (ICC); Murine organs, blood/human intestinal biopsies, intestinal organoids (small and large intestine); Staining techniques:Histo-pathological analysis of pro-inflammatory cytokines and epigenetic regulatory genes; proliferation; FISH (fluorescence in-situ hybridization; eubacterial 16S rRNA probes); evaluation of cell death/apoptosis; staining of stem cells and other epithelial/hematopoietic cell types; H&E (Hematoxylin and eosin) and PAS (Periodic acid Schiff-staining; to stain mucus producing goblet cells); Giemsa wright staining.
RT-PCR; RNA quality assurance by Bioanalyzer; RNA and DNA isolation of tissues/feces; Collecting and handling stool samples for 16s rRNA sequencing (for downstream metagenomics/metatranscriptomics analysis); Cloning (Transformation of E. Coli, DNA plasmid isolation, agarose gel electrophoresis, DNA extraction, restriction digestion, ligation & annealing); Genotyping and performing complex genetic breeding strategies (SPF and germ-free animals); Generating knockout mouse strains
INTESTINAL STEM CELL SYSTEMS
Human and mouse intestinal stem cell cultures (organoids)
Fluorescently label specific cell populations or bacteria
Protein electrophoresis and Western Blotting; Co-Immunoprecipitation; Nickel pull-down assay; Ubiquitin assay; Cytokine measurement by BioPlex Multianalyte Technology and ELISA; Co-Immunoprecipitation; Electro Mobility Shift Assay (EMSA); protein labeling; lipid droplet assays; subcellular fractionation assay