Improve Research Reproducibility A Bio-protocol resource
- Protocols
- Articles and Issues
- For Authors
- About
- Become a Reviewer
Categories
Animal model Antibody analysis
Complement analysis Host defense Immune cell differentiation Immune cell function Immune cell imaging Immune cell isolation Immune cell staining Immune mechanisms Immunotherapy Inflammatory disorder Mucosal immunologyEnvironmental Conditioning and Aerosol Infection of Mice
Influenza infection models in mice are widely used to study flu-mediated immune responses and pathology. However, most laboratory mice are housed at 20 °C and 50% relative humidity (RH). To better recapitulate influenza epidemics and immune responses during winter seasons, mice were housed at 20 °C under different humidity conditions, 10-20% or 50% RH. Here, we describe a protocol for using aerosolized droplets to infect mice with influenza under different environmental conditions. Using this method enables influenza infection studies performed under more physiologically relevant conditions which better mimics human viral exposure.
A Triple-challenge Mouse Model of Allergic Airway Disease, Primary Influenza Infection, and Secondary Bacterial Infection
Asthma is a global problem that affects millions of individuals. An increased risk of respiratory viral and bacterial infections is one of the complications of asthma. We recently reported that mice with ovalbumin-induced allergic airway disease (AAD) are protected against influenza-Streptococcus pneumoniae co-infection. Here, we describe in detail a protocol on how to induce AAD and influenza-S. pneumoniae co-infection in mice and to evaluate the specific roles of asthma on immunity to viral and bacterial pathogens in the hope of translating findings to benefit asthmatic individuals.
Quantitation of Regulatory Activity for the Complement Alternative Pathway Using an Adaptation of the AP50 in vitro Assay
Complement pathways function to identify and remove pathogens and infected cells. There are three complement pathways: the classical, lectin and alternative pathway (AP). While all pathways are activated following pathogen stimuli, the AP is constitutively active and tightly controlled by activators (e.g., Factor B, Factor D) and negative regulators (e.g., Factor H). Complement activity can be measured by well-established methods that are often used in a diagnostic setting to determine the CH50 (50% complement hemolytic activity) or AP50, specifically to measure AP activity. The protocol here has adapted the traditional AP50 method designed to measure AP activity in human sera, to measure the positive or negative AP regulatory activity within a given test sample. The assay relies on the ability of AP components in human serum to lyse rabbit erythrocytes under in vitro conditions specific for the AP with subsequent release of hemoglobin that is quantitated by measurement of optical density. Our method has added test substances, such as cell culture media with defined changes in individual complement components and determined the ability to either promote or inhibit AP activity in vitro. Thus, this protocol reflects the overall functional ability of a sample to effect AP activity and can be used in the research laboratory to determine AP regulatory activity in a complex biological sample, or to test the ability of drugs or novel biomolecules to regulate AP activity.
Virus Binding and Internalization Assay for Adeno-associated Virus
The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) based method to determine the amount of vector bound to the cell surface and the amount of subsequent virus internalization based on viral genome quantification. This protocol is optimized for studying AAV. Nevertheless, it can serve as a backbone for studying other viruses with careful modification.
Human Blood Component Vaccinia Virus Neutralization Assay
Many therapeutic viruses, such as oncolytic viruses, vaccines, or gene therapy vectors, may be administered by the intravenous route to maximize their delivery to target tissues. Blood components, such as antibody, complement and blood cells (such as neutrophils, monocytes, T cells, B cells or platelets) may result in viral neutralization and therefore reduce the therapeutic efficacy. This protocol will describe an in vitro assay by which to test the interaction of viruses with blood components. The effect of various factors can be isolated through fractionation. While whole blood can offer the most physiologically relevant snapshot, plasma can investigate the effects of antibody in concert with complement, and heat inactivated plasma will interrogate the effect of antibody alone.