Assay of Intracellular Free Calcium in Suspended B Cells Protocol This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+-
sensitive acidic form.
Assays for B Lymphocyte Function 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.
Characterization of Cells by Flow Cytometry Protocol Fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer.
Early Events in B Lymphocyte Activation Protocol 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.
Isolation of Resting B Lymphocytes from One or More Groups of Four Mouse Spleens Protocol Describes the isolation of resting B lymphocytes (B cells) from mouse spleens by using negative selection with anti-CD43 and anti-Mac-1/CD11b monoclonal antibodies coupled to magnetic microbeads. This strategy depletes non-B cells from a mixed population of splenocytes and relies on the fact that most mature leukocytes, with the exception of resting splenic B cells, express CD43.
Isolation of Resting B Lymphocytes from Sixteen Mouse Spleens Protocol Describes the isolation of resting B lymphocytes (B cells) from mouse spleens by using negative selection with anti-CD43 and anti-Mac-1/CD11b monoclonal antibodies coupled to magnetic microbeads. This strategy depletes non-B cells from a mixed population of splenocytes and relies on the fact that most mature leukocytes, with the exception of resting splenic B cells, express CD43.
Live Dead Assay for Cell Viability Protocol This assay is used to measure cell viability. It is a two-color fluorescence assay that simultaneously determines: Live cell number and Dead cell number.
Preparation of B-Lymphocyte RNA for Microarray Analysis Protocol 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.
Proliferative Assays for B Cell Function Protocol Describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen-induced proliferation) requires the laborious procedures of isolating antigen-specific B cells (which are otherwise present in too low a concentration in whole B cell populations).
Quantification of Apoptosis and Cell Cycle Distribution of Primary B Cells Using Propidium Iodide The water soluble, DNA intercalator, propidium iodide (PI), is used to bind to DNA after permeabilization of cells with NP40. The amount of dye bound correlates with the content of DNA within a given cell. Once cells are stained, they are analyzed on a flow cytometer. The relative content of DNA indicates the distribution of a population of cells throughout the cell cycle.
