Temporal Immune Profiling in the CSF and Blood of Patients with Aneurysmal Subarachnoid Hemorrhage.

Background: Delayed cerebral ischemia (DCI) is a significant complication of aneurysmal subarachnoid hemorrhage (aSAH). This study profiled immune responses after aSAH and evaluated their association with DCI onset.

Methods: Twelve aSAH patients were enrolled.

Neuronal binding by antibodies can be influenced by low pH stress during the isolation procedure.

Low pH stress and its influence on antibody binding is a common consideration among chemists, but is only recently emerging as a consideration in Immunological studies. Antibody characterizations in Multiple Sclerosis (MS), an autoimmune disease of the Central Nervous System (CNS) has revealed that antibodies in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis bind to myelin-related and non-myelin antigen targets. Many laboratories have used molecular biology techniques to generate recombinant human antibodies (rhAbs) expressed by individual B cells from healthy donors and patients with systemic autoimmune disease to identify antigen targets.

Co-culturing Immune Cells and Mouse-Derived Mixed Cortical Cultures with Oxygen-Glucose Deprivation to In Vitro Simulate Neuroinflammatory Interactions After Stroke.

Studying interactions between neural cells and glial cells in vitro remains an essential tool for scientists worldwide, and with the addition of oxygen-glucose deprivation (OGD) can be particularly useful for identifying mechanisms related to ischemic stroke-related injury and repair. In developing these protocols in the lab, however, we discovered the limitation of co-culturing immune cells with pure neuronal cultures as the standard media for immune cells impair neuronal growth and vice versa. Thus, we optimized a mixed cortical cell culture system that does not require the use of glial-conditioned media to support the viability and growth of neurons but can nonetheless be used to quantify neuronal survival and dendritic arborization.

A Guide on Analyzing Flow Cytometry Data Using Clustering Methods and Nonlinear Dimensionality Reduction (tSNE or UMAP).

Flow cytometry has been used for the last two decades to identify which immune cell subsets diapedese from the periphery into the brain parenchyma following injuries, including ischemic and hemorrhagic stroke. Recent developments have moved the analysis of high-parameter flow cytometry data sets from the traditional analysis method of manual gating to using unbiased analyses to improve scientific rigor. This chapter gives a step-by-step guide on using modern computational approaches to analyze complex flow cytometry data sets in FlowJo™ Software v10.

FACS to Identify Immune Subsets in Mouse Brain and Spleen.

Flow cytometry enables the multi-parametric quantification of cell types, especially in immunophenotyping of unique immune cell subsets that can either contribute to or ameliorate pathology. For tissues to be used in such analyses, single-cell suspensions must be created. Here we describe protocols for preparing single-cell suspensions of mouse spleen and brain tissue, as well as the steps for fluorescently activated cell staining/sorting (FACS).