lymphocyte Protocols

Category: Immunology: B Cell Protocols

Describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. - [Read Assays for B Lymphocyte Function Protocols]

Category: Immunology: B Cell Protocols

B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. Provides methods for the latter (direct) approach--namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II-antigen expression. - [Read Early Events in B Lymphocyte Activation Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

This method measures the leakage of DNA and lactate dehydrogenase from lymphocytes into the surrounding medium as an indicator of cytotoxicity.  This method also includes an assay of intracellular (mitochondrial) diaphorase as a measure of cellular activity (MTT assay). - [Read Human Lymphocyte Cytotoxicity Assay Protocol]

Category: Immunology: T Cell Protocols

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. - [Read Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol]

Category: Immunology: T Cell Protocols

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. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

Category: Immunology: T Cell Protocols

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. - [Read Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

Protocol describes a method for isolation and stimulation of lymphocytes. Leukocyte-rich plasma is collected from whole blood and then centrifuged through Ficoll-Hypaque. The collected cells are resuspended in growth medium containing various mitogens to stimulate growth. - [Read Lymphocyte Isolation and Culture Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Following propagation to 1 X 108 cells, lymphoblastoid cells are conveniently stored at -80 degrees C to preserve the high molecular weight DNA in the cells until the DNA is purified. This procedure describes the steps required to harvest and freeze the c - [Read Method: Preparation of Lymphocyte Cell Pellet for Storage]

Category: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression. - [Read Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte]

Category: Signalling Signal Transduction Protocols

This protocol provides a sufficient sample for several determinations of cAMP using the acetylation protocol. The method chosen for measuring the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) is an enzyme linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination]

Category: Signalling Signal Transduction Protocols

Protocol provides a method of achieving a sufficient sample for several determinations of cAMP using Amersham’s acetylation protocol. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination for Dual Ligand Screen]

Category: Immunology: B Cell Protocols

The method chosen for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) is an enzymelinked immunoassay system. Includes: Treatment of Cells and Preparation of Extracts. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination for Dual Ligand Screen Protocol]

Category: Immunology: B Cell Protocols

Procedure permits the isolation of at least 5 µg of total RNA from a sample of purified mouse splenic B lymphocytes. The quality of the RNA is assessed by separation of an aliquot through 1% agarose and staining with ethidium bromide as described in AfCS protocol Visualization of RNA Preparations on 1% Agarose Gels. The isolated RNA is used for analysis of gene expression by microarray technology. analysis of gene expression by microarray technology. - [Read Preparation of B-Lymphocyte RNA for Microarray Analysis Protocol]

Category: Microarray Protocols: Microarray Analysis Protocols

Protocol permits the isolation of at least 5 µg of total RNA from a sample of purified mouse splenic B lymphocytes Isolation of Resting B Lymphocytes from Sixteen Mouse Spleens. - [Read Preparation of B-Lymphocyte RNA for Microarray Analysis Protocol]

Category: Immunology: T Cell Protocols

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. - [Read Production of T Cell Clones Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes removal of T cell subsets by cytotoxic elimination using CD-specific antibodies. Describes the complete removal of T cells from lymphocyte preparations based on the presence of the glycoprotein Thy-1 on the cell surface of T lymphocytes. Cytotoxic elimination is employed; however, Thy-1-specific antibodies are used rather than MHC class II-specific antibodies so that T cells are eliminated rather than B cells and accessory cells. - [Read T Cell Depletion by Cytotoxic Elimination Protocol]