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.comFig. 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. 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.
Man’s immune system is subjected to a daily battle against invading pathogens and degenerate cells. A finely coordinated network of cells ensures that they are recognized and destroyed as early as possible. In this context one speaks of the two arms of the immune system: the innate immune system, made up of monocytes, macrophages, natural killer cells, granulocytes, and dendritic cells, and some others, and the adaptive immune system of T cells and B cells. The latter are necessary for building up a so-called immunological memory, so that the body is able to react and defend itself against a repeat infection of the same pathogens more quickly and powerfully.
 
Over the years scientists have identified an unbelievable number of proteins that are of diverse and significant importance to the cells of the immune system. They direct the targeted movement of and communication between the different cells for example. Other proteins recognize very specific parts of certain pathogens. These are taken up, broken down and finally presented to other immune cells. It has been shown that the various cells of the immune system can be identified and characterized by particular patterns of proteins that can be found on their surface. By means of these particular surface proteins it is possible to differentiate between T cells and dendritic cells for example in a blood or tissue sample. The established method of choice for this is flow cytometry. For this process, antibodies are used that very specifically recognize the appropriate surface proteins in a cell suspension (e.g. the protein Clec9A on a certain sub-group of dendritic cells). For visualization of the bonding, the antibodies are fluorochrome-conjugated (coupled with coloring agents). The colored mixture of single cells (cell suspension) is then scanned by the so-called flow cytometer. The process of hydrodynamic focusing facilitates the separation of the cells. With the aid of lasers, integrated in the flow cytometer, the antibody-fluorochrome complexes attached to the cells are energized and the resulting light that is emitted, is measured. Besides being used in flow cytometry, fluorochrome-conjugated antibodies are also employed in confocal immunofluorescence microscopy, where they facilitate the visualization of tissue structures and even single cells [1].

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.  

 
The Method
 
The flow cytometer used in this study (LSR Fortessa, Becton Dickinson), is equipped with two diode lasers (405 nm and 640 nm) and three solid state lasers (355 nm, 488 nm, and 561 nm), which the cells have to pass through one after the other with a minimal time delay of a few µs. This excites the fluorochromes coupled to the antibodies and which can be found on the surface of the cells. They then emit light at a wavelength which is very specific to them. These light signals are then passed through a fiber optic to complicated filtering optics where they are categorized.
 
Trigon or octagon technology is used here for each of the five lasers, in which up to respectively three or eight fluorochromes can be separately presented. So-called “Long-pass filters” only relay light signals of a clearly defined wavelength which are then additionally narrowed by a “bandpass filter” before they are finally registered by a detector, which then presents them as digital signals. Light of a shorter wavelength is reflected by the “Long-pass filters” and ultimately arrives at the next filter combination (fig. 1). Due to the lasers and filter systems employed in this flow cytometer it is possible to show 20 parameters in parallel. This includes the size, measured in the forward scatter channel (FSC), and the granularity of the cells, which is measured in the side scatter channel (SSC). The remaining 18 channels can be taken up by fluorochrome-conjugated antibodies or fluorescence dyes (e.g. DAPI) (fig. 2). 
 
The specific challenge in the work with a multi-parameter, flow cytometer lies in the choice and composition of these “antibody cocktails”. As when mixing a good drink, the individual ingredients are of ,significant importance. The choice of the appropriate fluorochromes also plays an essential role. All fluorochromes are characterized by their own particular excitation and emission wavelengths. As the range of the emission wavelength of one fluorochrome can partly overlap with another, it is important that they are compensated against each other before the experiment is started. In this case, for each fluorochrome used, a single coloration is used to determine whether it penetrates into the channel of another fluorochrome and can ultimately be recorded as a signal by the detector there. In a so-called compensation matrix all the fluorochromes used, are then adjusted to each other.
 
Figure 3 shows an example of a “gating strategy” of the human blood, with which samples are analyzed for the “patterns” of surface proteins described above. In this way the individual cell types of the immune system can be identified and further investigated. It was therefore possible for us to learn a lot about the biology of dendritic cells in human organs. Dendritic cells are also referred to as the “guardians of the immune system” and are located at the interface between the innate and the adaptive immune system. Extensive studies have shown that the dendritic cells in human blood, spleen, and thymus are very similar [2]. Another recently published study describes how, in the mouse model, specific immune responses can be triggered with the aid of certain surface proteins. These proteins (so-called Fc receptors or C-type lectin receptors) occurring on dendritic cells can be used as docking sites for antibodies. The latter carry pathogen-specific substances like in a backpack, which after binding to the docking sites are absorbed into the dendritic cells, whereupon specific immune reactions can be started [3].    
 
Conclusion
 
Flow cytometry presents an elementary method in immunological research. By simultaneously detecting 20 different parameters it is possible to specifically examine various cells of the immune system. This is of particular importance with regard to valuable or small samples.
 
Thanks 
 
We would like to thank the German Research Foundation (DFG) and the state of Bavaria for their financial support in the acquisition of an LSR Fortessa Flow Cytometer (INST 410/43-1 FUGG). In addition we thank the Bavarian Academy of Sciences, the Bavarian Genome Research Network, the SFB643 and the SFB1181 as well as the intramural support from the IZKF and the ELAN funds.

Authors:
Gordon F. Heidkamp1, Lukas Heger1, Christian H. K. Lehmann1, Diana Dudziak1

 
Affiliation
1University Hospital Erlangen, Department of Dermatology, Kussmaul Research Campus, Laboratory of Dendritic Cell Biology, Erlangen, Germany
 
Contact
Prof. Dr. Diana Dudziak
University Hospital Erlangen
Department of Dermatology 
Kussmaul Research Campus
Laboratory of Dendritic Cell Biology
Erlangen, Germany
diana.dudziak@uk-erlangen.de
 
 

References
[1] Eissing et al.: Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes, Immunology letters (2014)
[2] Heidkamp, Sander et al.: Human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment, Science Immunology (2016)
[3] Lehmann et al.: DC subset specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo, Journal of Experimental Medicine (2017)

 

Flow Cytometry - A Basic Introduction

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