A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehydeimethanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone 0, < 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde 0, < 0.006).With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37°C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors. 0 1992 Wiley-Liss, Inc.Key terms: Flow cytometry, cell fixation, membrane permeabilization, paraformaldehyde, methanol, multiparameter analysis, immunofluorescence There are many published studies in which specific intracellular proteins have been studied quantitatively by means of flow cytometric immunofluorescent techniques (1,(15)(16)(17)(18)(19)(20)(21)(22)(23)25), often in combination with cellular DNA content. Unfortunately, no clear consensus has emerged from these studies with regard to optimal cell fixation conditions for multiparameter flow cytometric analysis. In a number of studies, cells were fixed in methanol alone (8,15,19) or ethanol alone (1,3,7,9,10,16,18,25). However, comparative studies of alcohol with paraformaldehyde fixation have suggested that there may be significant loss of cytoplasmic or nuclear proteins from cells following fixation with alcohol alone (17,181.
Previous work from our laboratory had shown that goldfish retinal fragments explanted onto a polylysine substratum 1 to 2 weeks following optic nerve crush exhibit a striking clockwise pattern of neuritic outgrowth. In the present study, however, when the basal lamina component laminin was used as a substratum, neurites grew out as uncurved spokes, were less fasciculated, and had an increased rate of elongation. When laminin was combined with polylysine in the substratum, the degree of fasciculation and rate of elongation were similar to those seen on laminin alone, whereas the tendency for clockwise outgrowth was even more pronounced than that observed with polylysine alone. These results suggest that regenerating neurites have an affinity for laminin. Using an antibody to murine Engelbreth-Holm-Swarm sarcoma laminin, which cross-reacted with basal lamina in goldfish tissue sections, we studied the histochemical distribution of laminin in the goldfish visual system. Immunoperoxidase staining for laminin showed a characteristic scalloped pattern of staining in cross-sections of optic nerve bundles. Following optic nerve crush, the reaction product became much more diffuse and intense, especially distal to the crush site. When the retinal ganglion cell bodies were eliminated by removing the eye, the degenerating optic nerve stump still showed the intensive staining. We interpret these results to indicate that optic nerve glia are responsible in large part for the formation of laminin. Taken together, these in vivo and in vitro findings suggest that laminin plays a role in nerve regeneration in the goldfish central nervous system.
In order to examine the role of cell surface laminin in tumor metastasis we have utilized four well-characterized murine fibrosarcoma cell lines. Two of these lines were highly metastatic when injected into syngeneic mice while the remaining two lines were significantly less metastatic. Using indirect immunofluorescence techniques, we detected cell surface laminin on the cell surface of both highly metastatic cell lines but not on the low-metastatic cell lines. Although the low-metastatic cell lines did not possess endogeneous cell surface laminin, they had the ability to specifically bind exogenous laminin to their surface in a time- and concentration-dependent manner, indicating the presence of laminin receptors on these cells. Incubation of the low-metastatic cells with exogenous laminin prior to injection into syngeneic animals significantly increased their metastatic potential. No such increase was observed when the highly metastatic lines were preincubated with exogenous laminin. On the basis of these results, we conclude that in this fibrosarcoma model, metastatic potential is influenced by cell surface laminin and that the presence of unbound laminin receptors on the cell surface is not alone sufficient to promote metastasis of these cells.
Apolipoprotein A-I (apoA-I) is an important component of highdensity lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVAchallenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wildtype mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.
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