High-scoring articles simply do "mass spectrometry flow" technology dry goods

After 20 papers were published, the life of this female doctor after changing careers is even more exciting. A daily challenge for doctors: how to find a suitable cell sorting method! Six curves solved the difficulty of fitting 99% ELISA data. I asked my brother how to purify inclusion bodies, and he said ... Flow cytometry is widely used in many research fields and clinical diagnosis, especially in immunology, because of its advantages of multi-parameters, high resolution and rapid detection. The fly in the ointment is that traditional flow cytometry is easy to fall into the embarrassment of "blind people touching the elephant" when screening unknown cell groups. Mass spectrometry flow technology can solve this problem well. Through the leap of detection channel, more than 40 indexes can be detected at the same time, and a more comprehensive detection of cells can be realized. Like the traditional multi-color flow experiment, mass spectrometry also needs to consider the panel design, and be careful before the experiment. As the saying goes, a well-planned experimental scheme is the key to obtain effective data. Let's reveal the mass spectrometry process together. At the end of the article, there will be a CyTOF/Luminex multi-detection technology exchange meeting to experience the charm of mass spectrometry flow on the spot.

Want to know more about the principle of mass spectrometry shunt, please see: mass spectrometry shunt, the confession of a rich second generation.

Characteristic isotope labeling

The main difference between mass spectrometry and traditional flow technology is that metal elements and their isotopes are used as antibody markers. At present, rare earth elements, inert elements, late transition metals and halogens are mainly used as labels. The content of these elements in cells is very small, which will not cause high background and affect the normal physiological function of cells. At present, the main isotopes used as mass spectrometry process labels are shown in the following figure:

These labeled antibodies can be used to detect the expression and modification of cell-related biomarkers, cell activity, cell function and cell cycle.

Compared with traditional flow, the interference of adjacent channels in mass flow technology is much reduced, but overflow and leakage still exist, which are mainly caused by the following reasons:

(1). Isotope purity is not enough (impurity).

②.M 1 refers to the leakage of adjacent channels, which is called abundance sensitivity. Simply put, it is the peak tail with mass m and the signal interference with mass M+ 1 and m–1.

③.M+ 16 refers to the oxidation caused by isotope oxidation. For example, light lanthanides (139 ~ 160 amu) interfere with heavy lanthanides (155~ 176 AMU) due to the formation of oxides. At present, through the optimization of the experimental system, the interference of lanthanide isotopes which are most easily oxidized is reduced to less than 3.0%. 89y， 102pd， 104pd， 105pd， 106pd， 108pd， 1 10pd， 1 13in。

(1) Schematic diagram of interference signal source of mass spectrometry flow technology [1]

Chevrier S et al. use a single dye microsphere to calculate the leakage matrix between channels, which can be used as a reference for channel selection, as shown in Figure B:

(b) An overflow matrix calculated based on a single dyed bead. The diagonal value is 1. By default, only the leakage of potentially affected channels is calculated, including m 1, which corresponds to the known isotope purity of M+ 16. The number in the cell indicates the percentage of elements in the row leaking into the channel in the column. The number in the last column indicates the total amount of overflow and leakage signals received in the corresponding channel.

Basic strategy of panel design

Like the traditional flow, for the markers with different expression abundance, the principle of "strong and weak collocation" is followed, that is, for the markers with high expression abundance, isotope markers with low or medium sensitivity are selected; Isotopic markers with high sensitivity should be selected for markers with low expression abundance.

Antigens with low or unknown expression abundance:

Select high-sensitivity channels, such as153–176 ln and 209Bi；; Choose a channel that will not or rarely be interfered by other channels; It can be considered to further amplify the signal, such as labeling the same index with biotin secondary antibody labeled with metal elements or multiple antibodies with different clone numbers. Highly expressed antigen:

Select low sensitivity label (89y,102–10pd,13in, 1 15in) or medium sensitivity label (/kloc-0). Choose a channel that will not or rarely interfere with other channels; Optimize the antibody concentration and use the lowest antibody concentration that meets the demand, thus reducing the interference to other channels. In addition, you can follow the following rules:

1. mutually exclusive antigens (such as CD4 and CD8) can be arranged in the interference channel.

②. * * * expressed antigens (such as CD3 and CD4) are arranged in noninterference channels. If it is inevitable, the next stage (such as CD4) can be arranged to influence the upper channel (such as CD3).

③ Establish a perfect contrast. In addition to the necessary control in the experiment, a mass minus one (MMO) control, similar to the traditional FMO (fluorescence minus one control), can be set to judge the interference of the channel.

④ Distinguish single cell group from living cell group. Mass flowmeter has no FSC/SSC parameters to distinguish cells, so iridium or rhodium isotopes are often used as nucleic acid embedding agents to identify the whole cell population, and to distinguish adhesions and fragments according to DNA content. Cisplatin (platinum) is a chemotherapeutic drug, which can combine with the valence state of damaged cell membrane and is often used to distinguish dead cells from living cells.

Technical coffee face to face

The "20 19 CyTOF/Luminex Multi-detection Technology Exchange Conference" hosted by Bio-Techne will be held in Shenzhen. You are cordially invited to meet with experts of mass spectrometry and Luminex multiple detection technology to discuss their technical principles, experimental design and latest applications.

First, the style of the lecturer.

Professor Fu Guo graduated from Melbourne University, Australia. Former scientist of Scripps Institute, USA. Currently working in the School of Life Sciences, Xiamen University. His research interests are the function and development of immune cells, including the development, differentiation, function and related signal transduction of T cells and B cells, the interaction between pathogens and hosts, and the immune monitoring and treatment of tumors. Xiamen University has the first mass flowmeter in China, and Professor Fu Guo is one of the earliest academic leaders in China who are exposed to mass spectrometry flow technology. Rich experience and research data have been accumulated, and new reagents and schemes have been successfully developed on the mass spectrometry flow platform. Dr Guo, Ph.D., University of Melbourne, Australia, postdoctoral fellow, Commonwealth Scientific and Industrial Research Organization, Australia. He is currently the technical director of Guangzhou White Microbiology Technology Co., Ltd. From 20 to 0, 2005, he worked in the Commonwealth Scientific and Industrial Research Organization of Australia, engaged in the research on the effects of interferon and cytokines on the immune system and the corresponding gene regulation. 2010110/0 returned to China in October, and successively served as the project leader in Guangdong Veterinary Research Institute and Guangdong Laboratory Animal Monitoring Institute, engaged in animal health detection technology, especially the application of xMAP and xTAG technologies of Luminex to animal health detection. After returning home, * * * applied for 52 national invention patents, international PCT patents 1 1, authorized 32 national invention patents (including Luminex technology-related patents 17), and published 32 SCI papers (9 Luminex technology-related patents). Second, the timetable.

III. Time and place

Time: 2065438+May 26th, 2009 Location: Sheraton Shenzhen Bolin Hotel IV. Register for consultation.

Scan the QR code below to apply for registration; Or reply to the password "c0526" in the background of the official account of biologist WeChat to apply for registration. Tel: 02 1-52380373 Reference: [1]. Chevriers et al. cell system 2018may23; 6(5):6 12-620.e5. [2]。 Nat protocol. 2065 438+08 Oct； 13 (10): 2 121-2148. [3]. Biological macromolecules12,3997–401. [4].Behbehani GK, et al. Clinical Medical Laboratory. 20 17 years1February; 37(4):945-964.[5]. Laura T. Tang Lin, et al. Arthritis research. 20 18; 20: 139. article source: Bio-Techne image source: Bio-Techne, station cool Luohai title source: station cool Luohai image byGerd AltmannfromPixabay.

Subject: biotechnology, mutex antigen, * * * expressed antigen, dead cell, oxidative interference, spillover, characteristic isotope labeling, mass spectrometry, platinum, rhodium, iridium.