Multi-Parameter Flow Cytometry
Illuminating the Components of the Immune System
- Fig. 1: Octagon detection system for the violet (405 nm) laser. The light signals generated by the laser are passed through a glass fiber cable into the octagon system where they encounter a 690 nm “Long pass” filter (690LP). Only light at a wavelength of ≥ 690 nm can pass through this filter and in the next step a „Bandpass filter“ refines the selection even more. In this case using a 710/50 filter. Here, only light with a wavelength range of 685 nm to 735 nm is allowed through and recorded by detector A. Light signals of a wavelength shorter than 690 nm are reflected at the “Long pass” filter stage and encounter another combination of “Long pass” filter, “Band pass” filter, and detector. Figure adapted from: www.bdbiosciences.com
- Fig. 2: Schematic representation of diverse emission spectra. The LSR Fortessa flow cytometer has five lasers, three octagon, and two trigon filter optics for the simultaneous detection of up to 18 fluorescence signals and two morphological parameters (size and granularity). Here is an exemplary presentation of the excitation wavelengths of the different lasers, the available fluorochromes, their emission wave lengths, and the necessary filter systems. Figure: http://www.bdbiosciences.com/in/research/multicolor/ spectrum_viewer.
- Fig. 3: “Illuminating the components of the Immune System”. Using a multi-parameter flow cytometer, for example, it is possible to display different cell types of the immune system in parallel and to carry out interesting research on them. A sample “stained” with the fluorescent dye-labeled antibodies is measured with the flow cytometer, the resulting light signals are collected by the various detectors and finally displayed as digital image points. In the final step a so-called “Gating strategy” helps the scientist to pick out the important information from the entirety of the image points. In the example shown, three sub-groups of dendritic cells taken from human blood were examined. Here, it is necessary to include all immunological cells by means of the morphology gate (SSC-A / FSC-A). Subsequently, only single (FSC-H / FSC-A) and live (histogram, DAPI negative) cells are included in the analysis. As the sub-groups of dendritic cells only occur in low numbers, it is advisable to exclude other immune system cells from the analysis: B-cells (CD19/CD20 positive), T cells (CD3 positive), NK-cells (CD56/NKp46 positive) and Monocytes (CD14/CD11b positive). All dendritic cells are HLA-DR positive and, due to them having subgroup specific surface proteins, are sub-divided into plasmacytoid dendritic cells (pDC: CD123 and CD303 positive, blue box), CD1c+ dendritic cells (CD1c+ DC: CD11c and CD1c positive, red box) and CD141+ dendritic cells (CD141+ DC: CD11c and CD141 positive, green box). A comparable representation (Overlay- Histogram, figure below right) clearly shows that only one sub-group (CD141+ DCs, green) presents the protein Clec9A, on its surface. Cell parameters (FSC/SSC, Histogram Y-axis) are presented in linear scale and fluorescence parameters in logarithmic scale.
Previous models of flow cytometry were only able to present very few (max 4) separate fluorochromes. Accordingly, one was highly limited in simultaneous detection of certain cells. However over the last 10 – 15 years this technology has developed greatly, so that today, many more than four fluorochromes can be detected simultaneously.
Gordon F. Heidkamp1, Lukas Heger1, Christian H. K. Lehmann1, Diana Dudziak1
 Eissing et al.: Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes, Immunology letters (2014)
 Heidkamp, Sander et al.: Human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment, Science Immunology (2016)
 Lehmann et al.: DC subset specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo, Journal of Experimental Medicine (2017)