A novel microtiter plate assay for the quantitation of procoagulant activity on adherent monocytes, macrophage and endothelial cells.

The interaction of monocytes, macrophages and endothelial cells with inflammatory agents induces cell surface changes resulting in the activation of the coagulation cascade. A large volume 96 well plate microtiter assay has been developed which permits the quantitation of procoagulant activity on endotoxin stimulated cells without requiring the use of purified coagulation factors. Procoagulant activity is measured through a two stage amidolytic assay using commercially available Proplex as a source of factors VII and X and the chromogenic substrate S2222.

Synergistic effect of granulocyte-macrophage colony-stimulating factor and 1,25-dihydroxyvitamin D3 on the differentiation of the human monocytic cell line U937.

The human monoblastlike cell line U937 can be induced to differentiate by a variety of agents including gamma-interferon, phorbol esters, retinoic acid, and 1,25-dihydroxyvitamin D3 (VD3). Incubation of U937 with 1 to 1,000 units of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) did not induce macrophage differentiation. A synergistic effect on macrophage differentiation was observed, however, when U937 was cocultured with 10(-8) mol/L VD3 plus 50 U/mL GM-CSF.

Restoration of the lipopolysaccharide-responsive phenotype in C3H/HeJ peritoneal macrophage-P388D1 cell hybrids.

The fusion of thioglycollate-elicited peritoneal macrophages from the lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mouse strain to a hypoxanthine phosphoribosyltransferase (HPRT)-negative variant of the murine macrophage cell line P388D1 has resulted in the derivation of eight hybrid clones following HAT selection. Propidium-Iodide staining followed by flow cytometry has demonstrated that the DNA content of the hybrids represents the sum of the parents. Codominant expression of class I antigens from both parental haplotypes is observed in the hybrids.

Augmentation of procoagulant activity in monokine stimulated human endothelial cells by calmodulin/protein kinase C inhibitors.

The incubation of human umbilical cord endothelial cell cultures with inflammatory mediators results in the induction of procoagulant activity. As many of these mediators activate protein kinase C, the effect of calmodulin and protein kinase C inhibitors on IL-1, TNF, phorbol ester and LPS stimulated procoagulant activity was determined. Incubation of endothelial cell cultures with these inflammatory agents in the presence of phenothiazine derivatives or other classes of calmodulin and protein kinase C antagonists resulted in a 2-4 fold increase in procoagulant activity compared to parallel stimulated cultures in the absence of antagonists.

Simplified microELISA for the quantitation of murine serum amyloid A protein.

A simplified microELISA has been developed which permits the rapid quantitation of serum amyloid A protein, SAA, in acute-phase mouse sera. Serum samples are directly diluted without prior denaturation into bicarbonate buffer and coated overnight onto microtiter plates. A rat polyclonal anti-SAA serum is used as the primary antibody followed by an alkaline phosphatase-conjugated goat anti-rat IgG serum.