PubMed · 3721490
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H M Shapiro. 1986. Technical developments in flow cytometry.. https://doi.org/10.1016/s0046-8177(86)80174-5
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The ability of human peripheral blood mononuclear cells to respond to highly purified prothymosin alpha by generating superoxide anion was investigated. The generation of superoxide anion was detected by measuring the superoxide dismutase-inhibitable reduction of oxidized cytochrome C. Prothymosin alpha was shown to stimulate weakly these cells. The dose-response curve displayed a biphasic bell-shaped superoxide generation profile with two specific concentration optima for each individual blood donor, but with variations in optimal concentrations between the donors. By using a counter current centrifugation (elutriation) system, the mononuclear cell population was separated into several fractions according to their volume and density. Selective stimulation of these fractions with prothymosin alpha revealed that different cell populations were responsible for the generation of superoxide at higher and lower concentrations of stimulant, respectively. The response to the stimulus was immediate and lasted for a time period of about 4 to 8 min during which approximately 0.7 nmol O2- per min/10(6) cells were generated. The superoxide generation was cell-number-dependent with an optimum at 1 x 10(6) cells and lower rates for both smaller and larger cell numbers. Staurosporine, a potent inhibitor of protein kinase C, at concentrations sufficient to inhibit totally PMA-induced O2- generation, failed to affect the response of the cells to prothymosin alpha, while chelation of the extracellular Ca2+ abolished the lower but not the higher peak of O2- generation. Finally, simultaneous addition of prothymosin alpha and PMA resulted in a approximately 40% decrease of the O2- generation induced by PMA alone. A putative role as cell injury indicator is proposed for prothymosin alpha.
The most potent antigen presenting cell present in peripheral blood, lymphoid and non-lymphoid tissue is the dendritic cell (DC). The study of human DC has been restricted by their low frequency in the tissues and the lack of a truly DC specific surface marker to assist in identification and isolation. Standard techniques for the isolation of blood DC generally employ a period of in vitro culture followed by flotation on dense albumin gradients, or more recently, discontinuous gradients of metrizamide. Dense albumin gradients are time consuming to prepare, giving low and variable yields of DC. Metrizamide is more convenient, although exposure of monocytes to metrizamide can decrease the expression of CD14 and alter the accessory cell properties of antigen presenting cells. Here we demonstrate that Nycodenz gradient centrifugation of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclear cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are depleted and NK cell numbers are reduced two-fold from the interface cell population. The high density pellet fraction exhibits very little allostimulatory activity, indicating that few DC pass into the pellet. The low density fraction contains a significant population (20 +/- 5 (SD)%, n = 8) of cells which fail to stain for the lineage markers CD3, CD11b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does not alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.
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