Project description:The brain is difficult to analyze using flow cytometry because of its complex interactions of cells, high lipid content, and high autofluorescence. In this study, we investigated methods to isolate various types of brain cells with high yields and viability. The results showed that proteinase selection had a significant effect on the viability of various types of cells in the brain. The differences in the developmental stage also affected cell yield and viability. Moreover, the intensity of autofluorescence was found to differ greatly among the various regions of the brain. We also searched for neuronal markers that recognize a wide range of neurons. The ratios of the various exosomes contained by neurons differed depending on the type of neuronal marker. We also developed a method to quantify microglial phagocytosis in large quantities and in a short time using flow cytometry. These results have revealed critical factors that must be considered when analyzing various types of brain cells using flow cytometry.
Project description:This study was designed to investigate the efficacy of flow cytometry to accurately identify between normal and cancer cells in colon epithelium in humans diagnosed with colorectal cancer.
Project description:New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from twenty healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated good correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference dataset and highlights opportunities for further refinement. [Funding source] Project Number: 1ZIAHL006163-05 Contact PI / Project Leader: HOURIGAN, CHRISTOPHER Title: DETECTION, PREVENTION AND TREATMENT OF ACUTE MYELOID LEUKEMIA (AML) RELAPSE. Awardee Organization: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
Project description:To investigate a non-invasive strategy for immune monitoring the peripheral blood by flow cytometry, to address the critical need to itdentify predictive immunological biomarkers that correlate with treatment response Peripheral blood mononuclear cells (PBMCs) from 19 non–small-cell lung cancer (NSCLC) patients before and after ICI treatment and four healthy human donors were evaluated, utilizing spectral flow to monitor 24 immune cell markers simultaneously over the course of treatment. We performed immune cell profiling analysis using data obtained from RNA-seq of 19 different patients before and after immunotherapy, to validate the multi-color flow based immune profiling