Preparation of cells and reagents for flow cytometry.

Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies or ligands that bind to specific cell-associated molecules. A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.

Conjugation of fluorochromes to monoclonal antibodies.

UNLABELLED: Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is probably the most widely used application of flow cytometry. This unit contains protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, it provides a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. KEYWORDS: flow cytometry; monoclonal antibodies; fluorochromes; antibody labeling.

Characterization of a new antigen expressed by B and myeloid lineage cells identified by the monoclonal antibody LIP-6.

The LIP-6 MAb was produced against the undifferentiated cell line bh2-1 and recognizes an antigen expressed on all pre-B and B cell lines tested and some myeloid lineage lines. FACS analysis of normal tissues showed that LIP-6 is expressed on B lineage cells at all stages of differentiation, from bone marrow pre-B to plasma cells. T cells and thymocytes are LIP-6-, and splenic CD11b+ cells are heterogeneous for LIP-6 expression. The LIP-6 MAb was shown to precipitate a major 75-kDa and a minor 85-kDa protein under reducing conditions and a large protein of > 240 kDa under nonreducing conditions. Removal of N-linked sugars from the reduced lysates resulted in a single 65-kDa protein, suggesting that there is differential glycosylation of a single 65-kDa protein that forms disulfide-linked multimers. Finally, the LIP-6 antigen was shown not to be linked to the cell surface via a GPI linkage.

Flow cytometric detection of DNA tumor virus nuclear oncogene products in unfixed cells: saponin FACS of viral oncogene products.

Immunofluorescent analysis is a standard method for detecting DNA virus oncoproteins in transformed cells. Here we demonstrate the detection of DNA virus nuclear oncoproteins by flow cytometry of unfixed cells, after saponin permeabilization. This method could to be of value in the evaluation and quantitation of oncogene products in transformed cells.