Radioimmunoassay quantification of intra- and extracellular interleukin 1 alpha in murine macrophages, osteoblastic cell line and calvarial cultures (CROSBI ID 105847)
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Marušić, Ana ; Jastrzebski, Sandra ; Kalinowski, Judith ; Lorenzo, Joseph
engleski
Radioimmunoassay quantification of intra- and extracellular interleukin 1 alpha in murine macrophages, osteoblastic cell line and calvarial cultures
Interleukin 1 alpha and beta are two forms of interleukin 1 (IL-1) produced by a variety of activated cells, primarily by monocytes and macrophages.The development of specific sera and monoclonal antibodies made it possible to measure the forms of IL-1 with the use of specific radioimmuno (RIA) and enzyme-linked immunosorbent (ELISA) assays.In this report, we describe a construction of a simple RIA for mouse IL-1 alpha sensitive down to 50 pg/ml of IL-1 alpha.The assay was used to quantify the extracellular (EC) and intracellular-cell associated (IC) IL-1 alpha in murine macrophage cell line P388D1, spleen macrophages, calvarial tissue cultures and osteoblastic cell line MC3T3-E1.IL-1 alpha could not be detected in supernatants from unstimulated P388D1 and spleen cells.In contrast, there was 7- to 12-fold more IC IL-1 alpha in P388D1 cells.LPS increased the IC level of IL-1 alpha at a concentration as low as 1 ng/ml.Maximal increase occurred after g hours of lipopolysaccharide (LPS) stimulation.Immunoprecipitation of the freeze-thawed P388D1 cell extracts produced a single 17kD band that competed away with cold murine IL-1 alpha.IL-1 alpha could not be detected in conditioned media from calvarial cultures even after 10-fold concentration.However, LPS induced a dose-dependent increase of IL-1 alpha in calvarial freeze-thawed extarcts.Neither unstimulated nor LPS-stimulated MC3T3-E1 cells, amurine non-transformed cell line with osteoblastic phenotype, produced any detectable EC or IC IL-1 alpha.
Bone cells ; Interleukin 1 ; Macrophage ; Radioimmunoassay
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