The frequency of cells producing hemopoietic colony-stimulating factors (CSF) in a murine T lymphocyte clone has been determined by using a simple microassay that does not require clonal expansion or the addition of accessory cells. When stimulated with concanavalin A (Con A), the clone LB3 produced both granulocyte-macrophage CSF (GM-CSF) and multi-lineage CSF (Multi-CSF), which could be detected by using the cell line FDC-P1, whose proliferation is dependent on the presence of either of these factors. Limiting dilution analysis of Con A-stimulated LB3 cells indicated a requirement for cell-cell contact for optimal production of CSF, which could be bypassed by preincubation of the cells at high density with Con A for 4 hr before dilution in the assay. Limiting dilution estimates of the frequency of CSF-producing cells among Con A-pretreated LB3 cells ranged from 20 to 50%. Direct measurement of CSF production by single Con A-pretreated cells isolated by micromanipulation revealed that 10 to 20% could secrete detectable CSF. However, when isolated Con A-pretreated two-cell and three-cell aggregates were assayed, 50 to 99% were positive, indicating that 30 to 80% of the cells in the aggregates secreted CSF. Assay of the supernatants from single cells and two-cell aggregates on both FDC-P1 cells and another cell line, 32D c13, which responds only to Multi-CSF, demonstrated that many cells produced GM-CSF only, and others varied in the relative quantities of GM-CSF and Multi-CSF produced.

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