Servio Ramirez, Co-Investigator

The Cell and Immunology Core provides immunological expertise to explore neuroimmune interactions as related to substance abuse. The Core carries out assays of the effects of drugs of abuse on the immune system, using in vivo or in vitro assays. Many laboratories are interested in whether acute or chronic drug administration, or drug withdrawal or reinstatement, alter immune mediators in blood, particularly inflammatory molecules, or functioning of immune cells. These studies can be carried out on plasma, serum, cerebral spinal fluid, or cells from rodents, monkeys, or humans. A current major thrust of this Core is to explore the effects in the brain of molecules that have traditionally been thought of as immune mediators. Projects carried out in the Core have shown that chemokines are intimately involved in the neuronal actions of opioids and psychostimulants (cocaine and cathinones), including addiction, dependence and withdrawal, and the immune mediators are being measured in various brain regions after exposure to drugs of abuse. The imaging part of the Core allows investigators to assess neuronal and microglial receptor expression using immunofluorescence and confocal microscopy. The Core has new capabilities with the addition of a NIKON A1R+ Resonant Scanning confocal microscopy imaging system and the Nikon Elements AR software suite. This instrumentation enhances the capabilities of the system to enable 3D isometric rendering and volumetric quantitation to capture high resolution images of cells and molecular events with enhanced sensitivity.

Aim 1: Carry out assays of immune and physiological status including measurement of levels of cytokines, chemokines, and hormones in serum, brain dialysate fluid, or cell culture supernatants or in cells or tissue homogenates of brain or brain substructures, spleen, or lymph nodes using the following techniques

• The Magnetic Luminex Multiplex Array for quantitative measurement of levels of multiple cytokines, chemokines and other immune mediators in a single sample
• Enzyme ImmunoAssay (EIA-ELISA) to assess levels of individual immune mediators for individual molecules
• The Proteome Profiler Array (R&D Systems), a chemiluminscence Western blot-based technique used to screen for protein levels of panels of immune mediators
• The MSD Multiplex System with increased sensitivity for measuring individual mediators

Aim 2: To provide resources in histological methods, microscopy and image analysis

• For immunohistochemistry this includes: tissue processing, embedding, sectioning and detection using chromogens and fluorescence (single or multiplex). Advanced techniques offered include tissue clearing (via CLARITY) and special stains such Golgi-cox and Fluro-Jade staining.
• For detection of RNA in tissue, the core has available the in-situ hybridization platform from RNAscope (ACDbio)
• The Core also has available a comprehensive menu of microscopy and imaging capabilities such as: epifluorescence, confocal, intravital and live cell microscopy. Image analysis for area, particle analysis, tracking and 3D volumetric measurements can be performed using dedicated workstations configured to run NIS elements AR (Nikon) and Imaris (Bitplane).

Aim 3: To carry out functional immune assays

• Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ and skin graft rejection
• Other cellular assays such as response of lymphocytes to mitogens, Natural Killer cell activity, and in vitro antibody formation

Aim 4: To provide mycoplasma testing of cell lines for CSAR investigators