Summary:
At the protein level, flow has been the most commonly used single cell assay. However, due to problems such as cross-color, the flow can usually only detect 6 to 10 proteins simultaneously. The emergence of mass spectrometry technology has brought surprises to researchers. Using the superior resolution mass spectrometry technology and unique metal-labeled antibodies, the CyTOF2 mass spectrometry flow cytometer has improved both the number of channels and the signal quality.
Single-cell technology has developed rapidly in recent years. With the continuous development of single-cell sequencing, single-cell transcriptome and other technologies, humans have deep understanding of cell population heterogeneity from the level of DNA and RNA. At the protein level, flow has been the most commonly used single cell assay. However, due to problems such as cross-color, the flow can usually only detect 6 to 10 proteins simultaneously.
The emergence of mass spectrometry technology has brought surprises to researchers. Using ultra-high resolution mass spectrometry and unique metal-labeled antibodies, CyTOF2 mass spectrometry has a qualitative improvement in both channel number and signal quality.
Figure 1. Mass flow signal (right) has more channels and better signal resolution than traditional flow (left)
Compared to traditional flow, mass spectrometry has the following advantages:
First, the number of channels has increased to hundreds.
The ICP mass spectrometer in the mass spectrometry flow cytometer has a very wide atomic weight detection range (88 to 210 Da), so that hundreds of different parameters can be detected simultaneously.
Second, there is no interference between channels, no need to calculate compensation
ICP mass spectrometry has an ultra-high resolution that completely distinguishes the various elements used for labeling (as shown in the image to the right). This not only simplifies the experimental process, but also saves on specimens and reagents.
Third, the use of unique metal-labeled antibodies, the background is extremely low <br> using a new concept of metal-labeled antibodies, metal ions are covalently bound to the constant region of the antibody by polychelates. Since the cells themselves do not contain these metal elements as labels, there is no "auto-fluorescence" of the conventional flow type, and the signal background is extremely low.
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The sheer number of detection channels gives the mass spectrometry technology unprecedented data acquisition capabilities, allowing researchers to get more scientific information from a limited sample.
(A) analysis of bone marrow cells
Nolan Laboratories at Stanford University used a 31-channel assay to analyze bone marrow samples. The entire cell population was divided into approximately 30 subpopulations and analyzed for 18 signaling pathway molecules in each population.
Figure 2. To read cells' minds - mass spectrometry for detailed classification and signaling pathway analysis of bone marrow cells
(2) Analysis of T cell maturation process <br> More importantly, data mining with mass spectrometry streaming data combined with bioinformatics can help us understand the details of life processes and the heterogeneity of cell populations. For example, by processing the expression data of 25 surface and functional markers of T cells, the whole maturation process of T cells from the original Naive T cells to the effector T cells can be directly analyzed.
Figure 3. Analysis of T cell maturation process by mass spectrometry
(3) Drug screening <br> In addition, due to the variety of labels, the technology can be widely applied to various screening experiments, such as screening of antigenic determinants, drug screening and the like. It is also a powerful tool for single-cell proteomics research because of its powerful data acquisition capabilities.
Figure 4. Nature's cover article, drug screening through high-throughput flow analysis
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