| Autor: |
Page L; Institute for Laboratory Medicine and Microbiology, University Hospital Augsburg; lukas.page@uk-augsburg.de., Lauruschkat CD; Department of Internal Medicine II, University Hospital of Wuerzburg., Dennehy K; Institute for Laboratory Medicine and Microbiology, University Hospital Augsburg., Loell E; Institute for Laboratory Medicine and Microbiology, University Hospital Augsburg., Tappe B; Department of Internal Medicine II, University Hospital of Wuerzburg., Fuchs A; Internal Medicine III - Gastroenterology and Infectious Diseases, University Hospital Augsburg., Wurster S; Department of Infectious Diseases, Infection Control and Employee Health, MD Anderson Cancer Center, University of Texas Houston., Hoffmann R; Institute for Laboratory Medicine and Microbiology, University Hospital Augsburg. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2024 Sep 20 (211). Date of Electronic Publication: 2024 Sep 20. |
| DOI: |
10.3791/66593 |
| Abstrakt: |
Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 µL) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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