Soybean polypeptides and diisopropylfluorophosphate (DFP) have been reported to inhibit neutrophil functions such as the oxidative burst, chemotaxis, and/or phagocytosis in response to soluble stimuli; these observations would be compatible with the involvement of an active serine protease in neutrophil stimulation. We have investigated the possibility of such involvement when particulate stimuli such as immune complexes are utilized. The depolarization of the neutrophils' membrane potential, one of the earliest indicators of stimulation, and superoxide production, which is detectable 30-45 sec later, were our indicators of neutrophil response to immune complexes. The neutrophils were equilibrated with, and after 5 min washed free of, up to 60 mM DFP, a potent covalent serine protease inhibitor. At DFP concentrations below 24 mM, such treatment did not perturb neutrophil activation as measured by either of the above parameters, nor did F- alone under comparable conditions. Additionally, the immune complex induced responses of neutrophils preincubated for 3 min with N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK), L-1-tosylamido-2-phenyl-ethyl-chloromethylketone (TPCK), or phenyl-methyl-sulfonyl-fluoride (PMSF), covalent serine protease inhibitors which have, however, been shown to function in other capacities, e.g., as superoxide dismutases; 1 mM PMSF or 0.5 mM TLCK consistently reduced the observed membrane depolarization, one of the earliest consequences of neutrophil activation, by 20-30%, while 0.1 mM TLCK and 0.01 mM TPCK had little or no effect. The inhibition of superoxide production, a slightly later stimulus response, by PMSF, TLCK, and TPCK was more profound (50-75%); these compounds have, however, been shown to have activities other than serine protease inhibitors--for example, as superoxide dismutases. Since DFP is purely a serine protease inhibitor, and since the three other compounds do not affect depolarization (the earlier and superoxide independent event), our results indicate that active serine proteases do not appear to be necessary for immune-complex-initiated neutrophil stimulation. Other stimuli, which are known to activate neutrophils by different pathways, were not investigated.
Conventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization (MISH) method which facilitates hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosomes using hybridization rather than nucleic acid binding dyes. Key terms: GMD, flow cytometry, MISHGel microdrop (GMD) technology (33) evolved from an interest in encapsulating biological materials such as mammalian cells or microorganisms in agarose microspheres. Growth (25,34), secretion (25,26,34), metabolism (34,35,36), cytotoxicity (2,15,35), and electroporation (14) can be measured rapidly in the defined microdrop environment. GMDs are usually composed of agarose. They are prepared by dispersing molten agarose into an excess of a hydrophobic fluid, such as inert silicone oil, to form an emulsion. After the emulsion is transiently cooled, GMDs are separated from the silicone oil by centrifugation and remain physically distinct and robust.Various methods are available to analyze metaphase chromosomes, including flow cytometry (7,17,2 1 ), in situ hybridization (16,19,29,30,31), and staining (3,10). In order to provide stability, chromosomes are typically adsorbed onto slides for analysis. Consequently, analysis requires microscopic evaluation of individual slides which limits automation. Attempts to perform in situ hybridization on chromosomes in solution have been hindered by clumping, breakage, and aggregation ( 1 ). Current methods ( 18) could be improved by increased stabilization. Integration of GMD technology with hybridization techniques provides a system for analyzing chromosomes in suspension, facilitating sample handling, flow cytometry analysis, and hybridization-based isolation.GMDs provide assay microenvironments which are compatible with most in vitro cell manipulations. Our studies have indicated that GMDs can be pipetted, centrifuged, filtered, and analyzed by flow cytometry. The gel matrix provides physical protection, while the small size and porosity result in high permeability and rapid diffusion (35). These properties make the GMDs compatible with enzyme manipulation, fluorescence staining, and repeated washing.The results presented here demonstrate successful stabil ization and flow cytometric analysis of hybridized chromosomes. Development of this technology involved optimization of buffers, emulsion speeds, surfactants and agarose type. Successful encapsulation of chromosomes into GMDs was first confirmed using fluorescence microscopy and image analysis. Protocols were then developed for in situ ...
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