Since the 1970s, flow cytometry has been used in research and industry to characterize and sort cell populations. Cells can be analyzed and separated on the basis of their physical and molecular properties. The range of applications for this technique is very wide. In this blog article, we want to give an insight into various possible applications of flow cytometry using specific examples from research. Antibodies play a major role in flow cytometry, which should ideally be validated in particular for this technique.
These topics await you:
1) How Does Flow Cytometry Work?
2) Application Example 1: Isolation of Cancer-Associated Fibroblasts
3) Application Example 2: Isolation and Transplantation of Muscle Satellite Cells
4) Do FACS Analyses Affect the Gene Expression of the Investigated Cells?
How Does Flow Cytometry Work?
Flow cytometry (also known as FACS or “fluorescence-activated cell sorting”) enables the analysis, counting and sorting of cells in a fluid stream (Fig. 1). Heterogeneous cell populations can be separated based on their physical and molecular properties and analyzed down to the single-cell level in real time. This involves measuring more than 1000 cells per second. To use this technique, the cells to be analyzed must be in a single-cell suspension. They are then lined up one after the other as in a string of pearls in an extremely fine glass cuvette, so that each individual cell hits a laser placed behind the cuvette (xenon or argon lamps are also used). Each cell, that passes the laser beam, emits light that is measured by a detector and displayed on a computer in real time (Fig. 1). Depending on the properties of the cell, a specific optical signal is obtained and cells with different properties can be separated and documented.
As already mentioned, both physical and molecular properties can be measured. The measurable physical properties include: size, surface texture and granularity. Fluorescence-coupled antibodies recognizing intracellular or surface antigens are used to differentiate based on molecular markers. As with a fluorescence microscope, flow cytometry allows the sample to be excited with different wavelengths to separate and visualize labeled cells from unlabeled cells. A major advantage of this method is the ability to separate the cells based on their different optical signals and then collect them in separate reaction tubes for downstream applications. Subsequently, a wide variety of molecular biological investigations such as RNA/DNA analysis, proteomics or Western blot can be performed. It is also possible to put the isolated cells into cell culture.
Fig. 1: Flow Cytometry Instrumentation - How It Works. The workflow of flow cytometry starts with aspiration of the examined cells into the fluidic system (1). For the measurement, the cells are forced into a single file line (2), allowing the analysis on the single-cell level. Via a laser beam, the cells are excited, scatter the light and emit fluorescence depending on their individual properties (3). The fluorescence information is detected by different sensors, enabling the researcher to collect data about cell size, granularity and other properties (4). This data is then analysed to generate a comprehensive picture for each sample (5). Modified from [1].
Application Example 1: Isolation of Cancer-Associated Fibroblasts
The tumor stroma of many cancer types (especially breast cancer) contains cancer-associated fibroblasts (CAFs), which are often associated with a poor prognosis [2]. CAFs are an activated subpopulation of stromal fibroblasts, from which many express the myofibroblast marker α-SMA3. These fibroblasts are originated in the local tissue or bone marrow and become CAFs only under the influence of the tumor microenvironment, after being recruited into the tumor tissue. It has been shown that CAFs promote proliferation, angiogenesis and invasion of tumor cells. Working with CAFs is difficult because these cells exhibit tremendous heterogeneity with many subtypes in the tumor microenvironment. In addition, in vitro experiments with fibroblasts are complicated because they often change their expression profile in tissue culture. Neta Erez's research group has developed a protocol for isolating CAFs and normal fibroblasts directly from fresh tissue using fluorescence-activated cell sorting to circumvent these problems. To do this, they use antibodies against the fibroblast marker PDGFRα to label the desired cells. A single-cell suspension is prepared from the starting material, which is then incubated with the fluorescence-labeled PDGFRα antibody. Subsequently, PDGFRα-positive cells can be analyzed using a flow cytometer and sorted out for further analysis and characterization without tumor cell contamination [2].
Application Example 2: Isolation and Transplantation of Muscle Satellite Cells
When muscle tissue is injured, regeneration is mediated mainly by myoblasts. Their progenitor cells are muscle satellite cells. This stem cell population is normally mitotically quiescent in adult muscles. However, after injury, they initiate rapid cellular proliferation to produce myoblasts. Satellite cell-derived myoblasts are being investigated as a therapy for various regenerative diseases such as muscular dystrophy, heart failure, and urologic dysfunction. Transplantation of such cells into muscle fibers has demonstrated functional improvements in these diseases. In order to explore and perform this form of therapy, it is of great importance to establish efficient purification methods for quiescent satellite cells from skeletal muscles [3]. In 2005, Buckingham et al. developed a method to rapidly isolate this cell type for research [4]. Muscle satellite cells express the protein PAX3, by which they can be identified in skeletal muscles. Buckingham et al. used a PAX3(GFP/+) mouse line to sort satellite cells directly from tissue by FACS. The PAX3-positive cells fluoresce green without further staining or labeling steps and can thus be identified and sorted. The obtained cells were then transplanted into transgenic mice carrying a mutation in the mouse homolog of the human dystrophin gene (mdx gene for “muscular dystrophy x”). These mice serve as an established and internationally recognized model for Duchenne muscular dystrophy, in which severe muscle weakness occurs during life. The isolated and transplanted muscle satellite cells led to an improved fiber repair in the treated skeletal muscles of the mice [4]. Flow cytometry is thus also suitable to isolate cells for a medical/therapeutic purpose, as they do not lose their ability to divide or differentiate by this method and remain viable.
Do FACS Analyses Affect the Gene Expression of the Investigated Cells?
The method of flow cytometry as such offers a versatile tool for sorting and characterizing heterogeneous cell populations and can provide researchers with a great deal of information in a very short time. However, flow cytometry is often only the first stage and a means to an end, for example, to isolate one or more cell populations, which is often followed by further downstream applications. For some time now, gene expression analyses, for example using microarrays, have been of particular importance. Here, an important question arises: Does the analysis and sorting of cells by flow cytometry alter gene expression from the cells under investigation? Graham M. Richardson, Joanne Lannigan, and Ian G. Macara systematically investigated this question and published their results in 2015 [5]. They worked with mouse mammary gland tissue and isolated exemplary RNA after each experimental step up to sorting in different flow cytometers. Subsequent microarray analyses showed that the FACS analysis altered gene expression patterns of mammary gland cells to a very small extend. Only an upregulation of 18 miRNAs was observed, but this was caused by the previous steps of isolation. The authors concluded that FACS analysis had surprisingly little effects on the transcriptional patterns of the cells and that these effects can be further minimized by processing the cells constantly at 4°C [5].
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References:
[1] IDEX Health & Science, https://www.idex-hs.com/capabilities/life-science-optics/applications/flow-cytometry (last accessed 20.12.2023)
[2] Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS). J. Vis. Exp. (71), e4425, doi:10.3791/4425 (2013).
[3] Motohashi, N., Asakura, Y., Asakura, A. Isolation, Culture, and Transplantation of Muscle Satellite Cells. J. Vis. Exp. (86), e50846, doi:10.3791/50846 (2014)
[4] Margaret Buckingham et al., Direct isolation of satellite cells for skeletal muscle regeneration. Science, 2005 Sep 23;309(5743):2064-7. doi: 10.1126/science.1114758. Epub 2005 Sep 1.
[5] Graham M. Richardson,1 Joanne Lannigan,2 Ian G. Macara, Does FACS Perturb Gene Expression? Published online 13 January 2015 in Wiley Online Library, DOI: 10.1002/cyto.a.22608
