Human T Cell Activation Protocol Protocol for the Stimulation of human peripheral blood mononuclear cells with anti-human CD3 monoclonal antibody; MTT assay for detection of cellular proliferation. Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies. Performed titration studies with these antibodies and established the following protocol for stimulation of PBMC.
Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol Describes generating CTL against some commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions.
Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration.
Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes Protocol Peyer’s Patch, and Lamina Propria Cells lymphocyte populations should be analyzed when studying the immunological status of the intestine, for example in oral immunization or in intestinal disease (including infectious disease and tumors). This protocol details techniques for isolation of IEL, PP cells, and LP cells from the small intestine of the mouse.
MTT Assay for Detection of Cellular Proliferation Mouse Protocol: Stimulation of mouse peripheral T cells with plate-bound 145-2C11 monoclonal antibody;
MTT assay for detection of cellular proliferation.
Production of Mouse T Cell Hybridomas Protocol T cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). Protocol describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3-TCR complex by flow cytometry analysis.
Production of T Cell Clones Protocol Provides methods for the derivation of specific types of T cell clones: preparation and maintenance of alloreactive murine helper T (TH) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique and derivation of TH clones reactive with soluble protein antigens including a method for the selection of either TH1 or TH2 lymphocyte subsets.
Proliferative Assays for T Cell Function Protocols Protocols describe the conditions required to induce proliferation are described. Also describe the assay in which CD4·CD25·T cells are co-cultured with conventional T cells in order to assess their suppressive function. Will describe the culture conditions for the activation and expansion of CD4·CD25· cells.
Quantitation of Functional T Cells by Limiting Dilution Protocols In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2.
