PubMed · 6925903
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A Alonso Guerrero. 1982. [Hemotherapy].. https://pubmed.ncbi.nlm.nih.gov/6925903/
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A method for quantitating the proportion of cycling long-term culture-initiating cells (LTC-IC) in heterogeneous populations of human hematopoietic cells is described. This procedure involves incubating the cells of interest for 16 to 24 hours in a serum-free medium containing 100 ng/mL Steel factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF), with or without 20 microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating the recovered cells in standard LTC-IC assays. The details of this procedure are based in part on the finding that the number of LTC-IC (regardless of their cycling status) remains constant for at least 24 hours under these culture conditions, as long as 3H-Tdr is not present. In addition, we have determined that a 16-hour period of exposure to the 3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but not long enough to allow a detectable redistribution of LTC-IC between noncycling and cycling compartments. Finally, any isotope reutilization that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values measured. Application of this methodology to normally circulating LTC-IC showed these to be a primarily quiescent population. However, within 72 hours of incubation in a serum-free medium containing SF, IL-3, and G-CSF, most had entered S-phase, although there was no net change in their numbers. This suggests that, under certain conditions in vitro, self-renewal divisions of LTC-IC can occur and, at least initially, balance any losses of these cells due to their differentiation or death. In contrast, many of the LTC-IC in freshly aspirated samples of normal marrow were found to be proliferating, although those that were initially quiescent could also be recruited into S-phase within 72 hours in vitro when incubated under the same conditions used to stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further investigation of the mechanisms regulating the turnover of the most primitive compartments of human hematopoietic cells and how these may be altered in disease states or exploited for a variety of therapeutic applications.
IL-8 is a potent neutrophil chemoattractant that has been detected in high concentrations at acutely inflamed sites in vivo. Many cell types, including peripheral blood neutrophils, produce IL-8 that can be released by a variety of pro-inflammatory stimuli. However, the functional importance of neutrophil IL-8 during exudation is not yet known. We now report that neutrophils, harvested from skin lesions on the forearms of normal human volunteers (exudative neutrophils), expressed 100-fold higher levels of cell-associated IL-8 and spontaneously released up to 50-fold more IL-8 than freshly isolated peripheral blood neutrophils from the same donor. Furthermore, cell-associated IL-8 in peripheral blood neutrophils increased 20-fold during incubation at 37 degrees C in vitro and was increased over 200-fold after treatment with the Ca2+ ionophore A23187. More than 35% of the cell-associated IL-8 could be released by stimulation with either Ca2+ ionophore A23187 or phorbol myristate acetate. IL-8 was localized by sucrose gradient centrifugation to a subcellular fraction of heterogeneous, light membranous organelles. The accumulation of IL-8 within these organelles is inhibited by cycloheximide but not actinomycin D, suggesting that IL-8 accumulation is under translational, rather than transcriptional control. These studies indicate that peripheral blood neutrophils are capable of synthesis of large amounts of IL-8. Subsequent release of IL-8 during exudation may regulate neutrophil migration into sites of inflammation.
Fused silicon dioxide, multiparameter flow transducers with 50 microns internal square cross section and approximately 60 microns length can simultaneously measure DC and RF impedance as well as fluorescence and multiple-angle light scattering. A spherical version of such a transducer was mounted in an EPICS CVA flow-cell housing and was installed on a research prototype equipped with an argon-ion laser. The signal that was produced by the spherical transducer with EPICS DNA-Check beads was 1.73 times greater than that produced with the standard cylindrical flow cell. Similarly, with EPICS Immuno-Brite beads, the average ratio was 1.96. The Coulter impedance and light-scattering measurements were similar to those produced with the conventional cylindrical outside flow cell, although the internal cross section of the sphere was square and that of the cylinder was circular. The theoretical arguments of Leif and Wells have been demonstrated to be correct. At present, monolithic, spherical fused-silica transducers are the optimal design for combined electrooptical, multiparameter flow cytometry analyzers.