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Characterization and enumeration of haemopoietic cells grown in vitro is routinely performed either on unstained cultures or on cultures stained by cytochemical agents. This report describes a novel method for the immunocytochemical analysis of cells grown in plasma clots. The stain is performed in situ by subjecting the whole plasma clot in the culture dish to the staining procedure. The growth of early haemopoietic progenitor cells was monitored in cultures from normal human peripheral blood and proliferating progenitor cells were identified with an anti-human transferrin receptor monoclonal antibody followed by a two-layer immunoperoxidase stain. The number of transferrin receptor positive clusters demonstrable after 5 days of culture was similar to the number of haemopoietic colonies detectable after 12 days of culture.

Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay had the advantage of detecting only IL-2 with the ability to bind to the receptor, and displayed a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml.

We describe a bivariate flow cytometric assay to rapidly identify hybridomas producing new monoclonal antibodies recognizing subpopulations that are unreactive with existing immunological reagents. In this screen, whole cells in microtiter wells are labeled first with a red-linked test antibody, and then with a green-linked cocktail of existing monoclonal antibody reagents. The multiply-stained fluorescent cells are analyzed flow cytometrically and bivariate distributions of red vs. green-linked antibody fluorescence are generated. Test antibodies that recognize different subpopulations than those labeled by antibodies in the cocktail are readily identified. The use of an antibody cocktail conjugated with a single fluorophore allows comparison of the reactivity of the test antibody with multiple existing antibodies in a single analysis. This screen allows rapid (approximately 100 test antibodies can be evaluated in 40 min) identification of potentially interesting new antibodies for discrimination of subpopulations in heterogeneous tissues. We describe application of this assay to identify antibodies useful to mark hemopoietic subpopulations.

In order to facilitate the identification of human haemopoietic growth factors (HGFs), in complex conditioned media from limited cell sources, we have developed a method for the rapid assay of HGFs using bone marrow stem cells (BMSCs) obtained by Percoll density gradient enrichment of non-adherent cells from long-term human bone marrow culture. These BMSCs provide the basis for a 72 h assay where proliferation is triggered by the presence of GM-CSF, G-CSF and IL-3. The assay is up to 1000 times more sensitive than the conventional colony assay in detecting the presence of HGFs. We also describe methods for the rapid separation and identification of HGFs using fast protein liquid chromatography (FPLC) with phenyl-Superose and Mono Q columns. This enables individual HGFs present in complex conditioned media to be rapidly identified from the elution profile of HGF activity determined in the BMSC assay.

A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In this paper a method for the detection of the labeling index of normal and neoplastic colony-forming units (CFU) growing in plasma clot semisolid medium is described and preliminary results on the cell cycle of 7th and 14th CFU granulocyte-macrophage are discussed.

Cell surface receptors for immunoglobulin Fc regions can be detected by light microscopy of cytocentrifuge preparations of normal or malignant murine hematopoietic cells. The cells are first incubated in a culture supernate containing a monoclonal anti-hapten antibody, then with heat-killed, haptenated bacteria. Using 2 morphologically distinct strains of bacteria, we can detect both Fc receptors and cell surface differentiation antigens.