Comparison of Multiplexed Immunofluorescence Imaging to Chromogenic Immunohistochemistry of Skin Biomarkers in Response to Monkeypox Virus Infection.

Over the last 15 years, advances in immunofluorescence-imaging based cycling methods, antibody conjugation methods, and automated image processing have facilitated the development of a high-resolution, multiplexed tissue immunofluorescence (MxIF) method with single cell-level quantitation termed Cell DIVE. Originally developed for fixed oncology samples, here it was evaluated in highly fixed (up to 30 days), archived monkeypox virus-induced inflammatory skin lesions from a retrospective study in 11 rhesus monkeys to determine whether MxIF was comparable to manual H-scoring of chromogenic stains. Six protein markers related to immune and cellular response (CD68, CD3, Hsp70, Hsp90, ERK1/2, ERK1/2 pT202_pY204) were manually quantified (H-scores) by a pathologist from chromogenic IHC double stains on serial sections and compared to MxIF automated single cell quantification of the same markers that were multiplexed on a single tissue section.

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism.

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer.

Autofluorescence removal using a customized filter set.

Quantitative fluorescence microscopy is severely hindered by intrinsic autofluorescence (AF). Endogenous fluorescent molecules in tissue and cell samples emit fluorescence that often dominates signals from specific dyes. This makes AF removal critical to the development and practice of quantitative fluorescence microscopy.

Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity.

Doubly stochastic Poisson distribution of platelet adhesion on material surfaces and its implication on fluorescence image analysis.

An image based assay has been developed to quantify platelet adhesion on material surfaces. Briefly, citrated platelet rich plasma (PRP) is incubated with materials for 2 h to allow platelet adhesion on the surface, followed by fluorescence labeling of platelets with Celltracker Green. Multiple images are acquired by an automatic fluorescence microscope, IN Cell Analyzer 1000.

Shear stress regulates HUVEC hydraulic conductivity by occludin phosphorylation.

Human umbilical vein endothelial cells (HUVECs) display hydraulic conductivity (L(P)) responses to shear stress that differ markedly from the responses of bovine aortic endothelial cells (BAECs). In HUVECs, 5, 10, and 20 dyn cm(-2) steady shear stress transiently increased L(P) with a return to preshear baseline after a 2-h exposure to shear stress. Pure oscillatory shear stress of 0 +/- 20 dyn cm(-2) (mean+/-amplitude) had no effect on L(P), whereas superposition of oscillatory shear stress on steady shear stress suppressed the effect induced by steady shear stress alone.

A system for the direct co-culture of endothelium on smooth muscle cells.

The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function.

Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.

The objective of this study was to test whether a glycosaminoglycan component of the surface glycocalyx layer is a fluid shear stress sensor on endothelial cells (ECs). Because enhanced nitric oxide (NO) production in response to fluid shear stress is a characteristic and physiologically important response of ECs, we evaluated NOx (NO2- and NO3-) production in response to fluid shear stress after enzymatic removal of heparan sulfate, the dominant glycosaminoglycan of the EC glycocalyx, from cultured ECs. The significant NOx production induced by steady shear stress (20 dyne/cm2) was inhibited completely by pretreatment with 15 mU/mL heparinase III (E.

In vitro study of Starling's hypothesis in a cultured monolayer of bovine aortic endothelial cells.

Starling's hypothesis that fluid movement across the microvascular wall is determined by the transmural differences in hydrostatic and osmotic pressures was tested using an in vitro model comprised of bovine aortic endothelial cells grown on a porous support. In all experiments, a 1% bovine serum albumin (BSA) solution was maintained in the abluminal reservoir and the luminal reservoir contained either a 1 or a 5.5% BSA solution.