Performance of plate-based cytokine flow cytometry with automated data analysis

BACKGROUND: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. RESULTS: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. CONCLUSION: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses

The Interaction of Lingo-1 and Amyloid Precursor Protein

Thesis (Ph.D.)--University of Washington, 2012Proteolytic cleavage of amyloid precursor protein (APP) generates the amyloid β peptide (Aβ), the main component of cortical and subcortical plaques in Alzheimer's disease (AD). APP can be processed at the cell surface or within endosomes after endocytosis, and via an amyloidogenic or non-amyloidogenic pathway. Along the amyloidogenic pathway, APP is first cleaved by β-secretase followed by γ-secretase to produce Aβ. The non-amyloidogenic pathway involves cleavage by α-secretase and then γ-secretase. Aβ generation is thought to occur in a variety of organelles where APP, β- and γ-secretase reside. Proteins that regulate endocytosis and trafficking can thus control the qualitative proteolysis of APP, and consequently may be associated with pathophysiology of AD. One such protein is Lingo-1, which promotes APP trafficking to the lysosome and concomitant degradation, independent of the secretory pathway. In this manner, Lingo-1 may function as a control mechanism for APP levels

Developmental expression of paraoxonase 2

Paraoxonase 2 (PON2) is a member of the paraoxonase gene family also comprising PON1 and PON3. PON2 functions as a lactonase and exhibits anti-bacterial as well as antioxidant properties. At the cellular level, PON2 localizes to the mitochondrial and endoplasmic reticulum membranes where it scavenges reactive oxygen species. PON2 is of particular interest as it is the only paraoxonase expressed in brain tissue and appears to play a critical role in mitigating oxidative stress in the brain. The aim of this study was to investigate the expression of PON2 at the protein and mRNA level in the brain and liver of mice through development to identify potential age windows of susceptibility to oxidative stress, as well as to compare expression of hepatic PON2 to expression of PON1 and PON3. Overall, PON2 expression in the brain was lower in neonatal mice and increased with age up to postnatal day (PND) 21, with a significant decrease observed at PND 30 and 60. In contrast, the liver showed continuously increasing levels of PON2 with age, similar to the patterns of PON1 and PON3. PON2 protein levels were also investigated in brain samples from non-human primates, with PON2 increasing with age up to the infant stage and decreasing at the juvenile stage, mirroring the results observed in the mouse brain. These variable expression levels of PON2 suggest that neonatal and young adult animals may be more susceptible to neurological insult by oxidants due to lower levels of PON2 in the brain