Immune Systems and Components

In this video, we demonstrate the isolation of monocytes from whole human blood by immunomagnetic negative separation. The isolated monocytes can be used for further downstream analysis.

In this video, we demonstrate the isolation of Kupffer cells or liver macrophages from the mouse liver. The liver is perfused with a digestion solution followed by density gradient separation. The isolated adherent Kupffer cells can be used for further analysis.

This video demonstrates an enzyme-free protocol to isolate macrophages from mouse dorsal root ganglion (DRG) using a mechanical dissociation technique. Upon homogenization of the freshly collected DRG tissue, antibodies against the cell-surface receptors on the macrophages are used to label the cells fluorescently, followed by performing flow cytometry to confirm the presence of macrophages in the sample.

In this video, we demonstrate the isolation of lymphocytes from a harvested mouse Peyer's patch. The isolated lymphocytes can be used for further analysis.

In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further analysis.

In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

In this video, we demonstrate the isolation and culture of bone marrow stromal cells from the long bones of mice. The isolated adherent BMSCs can be used for further analysis.

This video demonstrates T cell isolation from peripheral blood mononuclear cells (PBMCs) using the functionalized buoyancy-activated cell sorting (BACS) technology. The isolation technique involves the positive selection of CD3+ T cells by labeling the cells with biotinylated anti-CD3 antibodies and separating the antibody-bound cells using streptavidin-bound microbubbles.

In this video, we describe a protocol for human primary T cell activation. The incubation of naïve T cells with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, followed by interleukin-2 or interleukin-15 treatment, leads to the activation of naïve T cells and their differentiation and proliferation into effector or memory T cells.

In this video, we demonstrate the isolation of marginating hepatic leukocytes from mouse liver by forced perfusion.

This video demonstrates the isolation of lymphocytes, including natural killer cells or NK cells, from human liver tissue for selective expansion. Enzymatic and mechanical digestion, followed by density gradient separation, promotes lymphocyte isolation, including NK cells. The collected lymphocytes can be further used for the selective expansion of NK cells.

This video demonstrates a technique for the expansion of natural killer (NK) cells directly from peripheral blood mononuclear cells (PBMCs) using a feeder cell line. PBMCs are co-cultured with genetically modified lymphoblastoid feeder cells expressing membrane-bound interleukin-21 (mIL-21), and the culture is supplemented with the cytokines interleukin-2 (IL-2) and interleukin-15 (IL-15). Together, this cocktail of ligands leads to the prolonged expansion of NK cells with high purity.

This video demonstrates the isolation procedure of Sertoli cells from mouse seminiferous tubules, a component of the testes. Enzymatic digestion and repeated washes promote the release of Sertoli cells into the solution, which can be cultured further for proliferation and growth.

This video demonstrates a protocol for the magnetic labeling-based isolation of a low-concentration population of CD4+Vδ1+γδ T cells from human peripheral blood mononuclear cells (PBMCs). Using a combination of FITC-conjugated anti-Vδ1 antibodies, magnetic anti-FITC microbeads, and anti-CD4 antibody-coated superparamagnetic beads, the CD4+Vδ1+ T-cells can be successfully purified from the PBMC population.

In this video, we demonstrate the isolation of cytotoxic high-density neutrophils and low-density neutrophils from the blood of a tumor-bearing mouse by discontinuous sucrose gradient centrifugation.

This video demonstrates the isolation procedure of the gingival immune cell network from a mouse model. The enzymatic digestion releases the gingival tissue from the attached teeth and bone, which is further mechanically digested, releasing immune cells from the gingiva. The isolated cells are ready for immunological experiments.

This video demonstrates a peritoneal lavage method for isolating mature connective tissue-type mast cells from the peritoneal cavity of adult mice. Upon isolating a mixture of primary immune cells from the peritoneal cavity, selective proliferation of mast cells is performed under appropriate culture conditions.

In this video, we describe a protocol for isolating CD11b cells, including microglia, from mouse pup brains using magnetic-activated cell sorting. The brain tissues are homogenized to obtain single-cell suspensions, which are incubated with anti-CD11b antibody-coated microbeads, followed by a magnetic column-based separation to purify the CD11b+ cell population.

This video demonstrates the surgical isolation procedure of the inguinal draining lymph node from a mouse model. Incision of the abdominal region provides access to the inguinal lymph node, which is surgically harvested. The harvested translucent inguinal lymph node can be used for immunological studies.

This video demonstrates a dissection technique to harvest popliteal draining lymph nodes from the mouse hind legs. Lymph nodes play a critical role in bridging innate and adaptive immunity, and harvesting intact lymph nodes — with a preserved tissue structure and cellular viability — can help study local and systemic immune responses.

This video demonstrates the priming of primary monocytes into macrophages. Primary human monocytes can be isolated and differentiated to an M1-like pro-inflammatory phenotype using the granulocyte-macrophage colony-stimulating factor (GM-CSF) or to an M2-like anti-inflammatory phenotype using the macrophage colony-stimulating factor (M-CSF).

This video describes an in vitro method for generating macrophages from human induced pluripotent stem cells (iPSCs). Macrophage generation from iPSCs occurs in three stages: (i) aggregation of iPSCs in suspension to form three-dimensional embryoid bodies (EBs) that differentiate into mesodermal cells, (ii) formation of macrophage precursor cells from the differentiated EBs, and (iii) differentiation of the macrophage precursor cells into mature, functional macrophages.

In this video, we demonstrate a procedure to induce the differentiation and polarization of peripheral blood-derived monocytes into M1 or M2 macrophages. Incubation with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and interferon‐gamma yields pro-inflammatory M1 macrophages, while incubation with macrophage colony-stimulating factor and interleukin-4 induces anti-inflammatory M2 macrophage production.

This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with γ-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.

This video demonstrates the differentiation of bone marrow stromal cells (BMSCs) into adipocytes. In the two-step process, in the presence of specific growth factors and hormones, undifferentiated BMSCs first differentiate into preadipocytes. The second step involving treatment with glucose and insulin leads to the formation of mature adipocytes with multiple lipid droplets.

This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells — isolated from human umbilical cord blood — differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

This video demonstrates a protocol for generating functional antigen-specific T cells from iPSC-derived immature T cells using a three-dimensional thymic culture system. Co-culturing the iPSC-derived immature T cells with endogenous lymphocyte-depleted mice fetal thymic lobes resulted in successful maturation of the T cells to form CD8αβ+ MHC class I+ iPSC-derived thymic emigrants (iTEs).

In this video, we describe a protocol for purifying platelets from murine whole blood and their subsequent activation with the platelet agonist thrombin. When analyzed with flow cytometry, the purified resting platelets are characterized based on the expression of their cell-surface marker CD41, whereas purified activated platelets co-express both the surface markers CD41 and CD62P.

This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.

This video demonstrates the in vitro differentiation of granulocyte-macrophage-colony-stimulating factor or GM-CSF-producing T helper cells (THGM). Isolated naïve T cells are stimulated for differentiation into THGM, which is confirmed by detecting high intracellular GM-CSF production and a low expression of other T helper cell-specific cytokines.

This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.

In this video, we describe a protocol for the flow cytometry-based sorting of naïve B cell, memory B cell, and plasmablast plasma cell subpopulations from human peripheral blood B cells. The different B cell subpopulations can be distinguished based on fluorophore-based antibody binding to the CD19, CD27, and CD38 receptors expressed on the B cell surfaces, followed by cell sorting.

This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody's isotype, which correlates with the B cells' secretory activity.

In this video, we describe a protocol for isolating single cells from murine pancreatic draining lymph nodes (PDLNs) and the characterization of regulatory T cells (Tregs) from the single-cell population using flow cytometry. Tregs are identified based on the positive expression of the cell-surface markers CD4, CD25, and Nrp1, along with the nuclear transcription factors Foxp3 and Helios.

This video demonstrates the in vitro study of immunological synapse formation between an antigen-presenting cell and helper T lymphocytes using a fluorescence microscope. T cell receptors of helper T lymphocytes interact with an antigen-MHC-II complex in antigen-presenting cells and form immunological synapses, followed by secretory vesicle movements toward the synapse, which is visualized by fluorescent microscopy.

In this video, we demonstrate a flow cytometry-based technique to determine the proportion of cytokine-induced killer T-cells, CIK cells, expanded from a peripheral blood mononuclear cell culture.

This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-γ) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-γ accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-γ inside the cells.

In this video, we describe a protocol for the immunostaining and confocal microscopy-based visualization of macrophage extracellular traps (METs) in murine lung tissue. Tissue-resident macrophages are characterized by the expression of the macrophage-specific F4/80 surface glycoprotein marker, while METs are visualized by labeling the MET markers matrix metalloproteinase 9, citrullinated histone H3, and extracellular chromatin.

This video demonstrates an efficient homogenization technique to generate an antigen-adjuvant emulsion to induce experimental autoimmune encephalomyelitis (EAE) in a murine model. The emulsion contains an autoantigen — myelin oligodendrocyte glycoprotein (MOG) — in the water phase, surrounded by a continuous oil phase comprising an inactivated pathogenic bacteria. The emulsion ensures a sustained release of antigens for a prolonged immune response.

This video demonstrates the visualization of γδ intraepithelial lymphocytes (IELs) in transgenic mice using confocal microscopy. The surgical procedure involves creating a perforation line and making a longitudinal incision to expose the intestinal mucosa and villi. The transgenic mice express reporter proteins exclusively in γδ IELs, allowing their tracking. Intravital imaging captures the dynamic movement of green fluorescence-labeled γδ IELs along the intestinal epithelial cell's basement...

This video demonstrates the generation and isolation of T lymphocyte-derived microparticles, LMPs, from T lymphocytes following treatment with actinomycin-D.

This video describes a protocol for isolating rare-antigen-specific CD8+ T cells using magnetic nanoparticle-based artificial antigen-presenting cells (aAPCs) functionalized with antigenic peptide-loaded MHC-Igs and anti-CD28 antibodies. Interaction of the aAPCs with CD8+ T cells specifically expressing the antigenic peptide-targeting T cell receptors, followed by their expansion in vitro, results in the enrichment and expansion of this CD8+ T cell population.

This video demonstrates separating neutrophils from tracheal secretions using a microfluidic device with spiral channels. The device achieves efficient separation based on cell size by utilizing varying thicknesses and trapezoidal-shaped channels. The interplay between forces results in large neutrophils accumulating near the inner wall while erythrocytes and mucin aggregates remain near the outer wall, ensuring effective neutrophil separation.

In this video, we demonstrate the intracellular radiolabeling of T helper cells using radioactive isotope-conjugated monoclonal antibodies for non-invasive positron emission tomography imaging.