Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry

Journal article


Publication Details

Author(s): Fuernrohr BG, Stein M, Rhodes B, Chana PS, Schett G, Vyse TJ, Herrmann M, Mielenz D
Journal: Journal of Immunology
Publication year: 2015
Volume: 195
Journal issue: 1
Pages range: 377-85
ISSN: 0022-1767


Abstract


Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.



FAU Authors / FAU Editors

Herrmann, Martin Prof. Dr.
Medizinische Klinik 3 - Rheumatologie und Immunologie
Mielenz, Dirk Prof. Dr.
Medizinische Fakultät
Schett, Georg Prof. Dr. med.
Lehrstuhl für Innere Medizin III
Stein, Merle
Professur für Immunologie


External institutions with authors

Guy's and St Thomas'


How to cite

APA:
Fuernrohr, B.G., Stein, M., Rhodes, B., Chana, P.S., Schett, G., Vyse, T.J.,... Mielenz, D. (2015). Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry. Journal of Immunology, 195(1), 377-85. https://dx.doi.org/10.4049/jimmunol.1401889

MLA:
Fuernrohr, Barbara G., et al. "Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry." Journal of Immunology 195.1 (2015): 377-85.

BibTeX: 

Last updated on 2019-18-07 at 07:30