Abstract: A method and apparatus for preparing biological cell samples for intracellular analysis. The invention is based upon the recognition that many of the steps of the conventional methods for such sample preparation can be eliminated, leading to a process that readily lends itself to automation and the advantages associated therewith. The method of the invention comprises the steps of (a) cell-fixation, (b) permeabilization and (c) staining (or labeling) of intracellular molecules of interest by probes that are readily detectable by flow cytometric techniques, all without any intervening cell-washing (and re-suspension) steps. Rather, the single cell-washing step is effected after these three steps have been carried out. Preferably, the washing step is carried out by passing the fixed, permeabilized and stained cell sample through a semi-permeable membrane that serves to filter out (by transmission) interferants to waste while retaining the cells of interest.

Abstract: A method and apparatus for preparing biological cell samples for intracellular analysis. The invention is based upon the recognition that many of the steps of the conventional methods for such sample preparation can be eliminated, leading to a process that readily lends itself to automation and the advantages associated therewith. The method of the invention comprises the steps of (a) cell-fixation, (b) permeabilization and (c) staining (or labeling) of intracellular molecules of interest by probes that are readily detectable by flow cytometric techniques, all without any intervening cell-washing (and re-suspension) steps. Rather, the single cell-washing step is effected after these three steps have been carried out. Preferably, the washing step is carried out by passing the fixed, permeabilized and stained cell sample through a semi-permeable membrane that serves to filter out (by transmission) interferants to waste while retaining the cells of interest.

Abstract: Ligand-aminodextran-(phycobiliprotein or tandem dye) conjugates useful for detection of a desired target biological material by providing an enhanced fluorescent signal are described. Also described is a method for a single-measurement quantification of multiple populations of cells based upon the labeling of different pairs of cell populations, each pair containing mutually exclusive cell receptors which are expressed at substantially similar receptor densities with labeled ligands for each receptor. One cell population is labeled with a ligand capable of binding to a first cell surface receptor which ligand is directly conjugated to a fluorescent phycobiliprotein or tandem dye; and a second cell population is labeled with a ligand capable of binding to a second cell surface receptor, which ligand is cross-linked to an aminodextran to a fluorescent phycobiliprotein or tandem dye.

Abstract: A method for a single-measurement quantification of multiple populations of white blood cells (WBC) is based upon the labeling of different pairs of cell populations, each pair containing mutually exclusive cell receptors which are expressed at substantially similar receptor densities with labeled ligands for each receptor. One cell population is labeled with a ligand capable of binding to a first cell surface receptor which ligand is directly conjugated to a fluorescent phycobiliprotein or tandem dye; and a second cell population is labeled with a ligand capable of binding to a second cell surface receptor, which ligand is cross-linked by an aminodextran to a fluorescent phycobiliprotein or tandem dye. The phycobiliproteins upon laser excitation produce a different detectable fluorescence intensity for each cell population. Use of such pairs of conjugates enable two populations of cells with similar receptor densities to be distinguished with the use of a single color marker.