Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays.

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays.

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo.

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.

Combining ELISPOT and ELISA to measure amounts of cytokines secreted by a single cell.

Enzyme-linked immunospot (ELISPOT) assay allows for the determination of the frequency of -cytokine-secreting cells, but does not answer the question of how much cytokine is secreted per cell. In our study, we combined ELISPOT and ELISA assays and developed a protocol to calculate the amount of IFN gamma secreted by each cell. A suspension of human peripheral blood mononuclear cells was split into two pools and cells from one pool were cultured in a regular ELISPOT plate, whereas cells from the other pool were cultured in an uncoated, "blank," ELISPOT plate.

ELISPOT assay for neuroscience research: studying TNFα secretion from microglial cells.

The major application of ELISPOT assays is to study secretion of cytokines and chemokines from immune system cells. We adapted this assay to study TNFα secretion from microglial BV2 cells, which are similar in physiology to microglia in the nervous system. Stimulation of BV2 cells with 1 μg/mL LPS resulted in a robust secretion of TNFα.

ELISPOT assay as a tool to study oxidative stress in lymphocytes.

Enzyme-linked immuno spot (ELISPOT) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of ELISPOT assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. We worked out a protocol to study oxidative stress in human peripheral blood lymphocytes by determining their potency to secrete IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha in response to acute treatment with hydrogen peroxide.

Equine ELISPOT assay to study secretion of IFNγ and IL-4 from peripheral blood mononuclear cells.

Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA).

ELISpot assay as a tool to study oxidative stress in peripheral blood mononuclear cells.

Enzyme-Linked Immuno Spot (ELISpot) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of an ELISpot assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell.We have shown that hydrogen peroxide-induced oxidative stress was causing ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokine IL-8 and cytokine TNF alpha.

Roundabout 4 is expressed on hematopoietic stem cells and potentially involved in the niche-mediated regulation of the side population phenotype.

Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation.

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

Unlabelled: RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool.

The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity.

NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules.

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells.