The immune system is like the body's guardian, silently guarding our health. The immune cells that make up this escort team are responsible for removing bacteria, viruses, and foreign bodiesOn the other hand, it is the "best doctor of the human body" by eliminating senescent cells and mutated cells in the body, laying the foundation for health.
Figure 1Members of the immune system.
NK cells, T cells, B cells, etc., are all members of the immune system, which we introduced in the previous flow cytometry classth1/th2/th17withtregThe selection of cells and their indicators will be briefly introduced to you todayNK cellsand explain its detection indicators and case analysis.
Related reading: elabscience flow cytometry classroom th1 th2 th17 cell introduction and index selection elabscience flow cytometry classroom |Introduction to TREG cells and selection of indicators
Introduction to NK cells
NK cells are a member of lymphocytes, also known as natural killer cells (NK cells), which are important immune cells in the body. NK cells are morphologically large granular lymphocytes, the third largest type of lymphocytes in addition to T cells and B cells, accounting for about 15% of all immune cells (white blood cells) in the blood, and belong to the core cells of the innate immune system, mainly distributed in the peripheral blood, liver and spleen. itIt can destroy target cells without antigen presensitization, and it is non-mhc restrictive, and the response speed is fast, which can play a role in the early stage of the immune response
Figure 2Schematic diagram of NK cells.
NK cells are the most important "warriors" in the body responsible for killing abnormal cells such as aging, viral infection, and tumors. In addition, in the face of cancer cells, NK cells do not need the activation and sensitization of other cells to directly recognize and kill cancer cells, so they are also known as the first line of defense against cancer in the human body. If the number of NK cells decreases or their function decreases, the chance of developing neoplastic diseases is significantly higher.
Compared with neutrophils, macrophages, etc., which also belong to the same family of innate immune cells, itsThe range of bacteria and viruses is wider
In contrast to the acquired immune cell T cells, it is in anticipating the invasion of the "enemy".It is more responsive and has a wider spectrum of "kills".It is not only limited to killing tumor cells, but also has made achievements in resisting viruses and anti-aging.
Development and classification of NK cells
At present, the mainstream NK cell development model believes thatNK cells** in CD34 + CD45Ra+ hematopoietic progenitor cells (HPCs) in the bone marrow。In the bone marrow, NK cells develop from HPCs through common lymphoid progenitor cells (CLPS) and NK cell precursors (NKPs), and then migrate to peripheral blood (Conventional NK cells) or tissue-resident NK cells (TRNK cells).
The differentiation of TRNK cells occurs at different tissue sites, including the lungs, thymus, liver, uterus, subcutaneous adipose tissue, and kidneys. At these sites, NK cells have different phenotypic characteristics and functions that make up the cycle of NK cells at different stages of maturation. In addition to different tissue types,Even within the same organ and the same tissue, NK cells are highly heterogeneous
Figure 3Development and subpopulations of NK cells.
NK cells are often defined as CD3 CD56+ cells and are usually divided into two categories based on the relative expression of the surface proteins CD56 and CD16:cd56hicd16-(immunomodulation, cytokine production) andcd56lowcd16+(cytotoxicity). Among them, CD56lowCD16+ accounted for 90% of all NK cells, and CD56hiCD16- low accounted for only about 10%.
NK cell assay index selection and cases
How do you choose an indicator to detect NK cells?Based on the past experimental experience, the following summary is made for everyone:
human：cd3-cd56+cd16+
mouse：cd3-cd49b+/nk1.1+
A case of human peripheral blood NK cell detection
Human NK cells express the specific indicator CD56 during development, and some NK cells also express FC RIIIA (CD16), and CD57 at the end of development. Samples of peripheral blood or other non-purified NK cells should be tested with CD3 to exclude a special type of T cell, NKT (CD3+CD56+). The results of human peripheral blood NK cells are shown in the figure below
Figure 4Flow cytometry results of human peripheral blood NK cell detection.
The criteria for the detection of NK cells are:cd3-cd16+cd56+
CD16 and CD56 should preferably have an isotype control, other indicators can be omitted due to the obvious clustering.
Human peripheral blood samples are recommended to be stained with CD45By CD45 with SSC, it is beneficial for lymphocyte gating。CD45 can be used as a single stain tube, at low speed, and the threshold can be set.
Case of detection of Nk cells in C57BL 6 mice
CD49b and NK11 is the two markers for the identification of mouse NK cells, and one of them needs to be selected according to the mouse strain during the experiment. For example, CD49B (DX5) is used for BALB C mice and NK1 is used for C57BL6 mice1. Analyze the time circle CD3-CD49B+ or CD3-NK11+。The results of C57BL6 mouse NK cells are shown in the figure below
Figure 5Flow cytometry results of C57BL6 mouse NK cell assay.
Peripheral blood samples increased CD45 index, directlyLymphocyte populations are circled by CD45 and SSC
The detection criteria for C57BL6 mouse NK cells are:cd3-nk1.1+
The experimentalThe key factor is red blood cell lysis, excessive or insufficient lysis of red blood cells can result in inconspicuous lymphocyte population segmentation.
NK cell assay flow cytometry antibody recommended
NK cell assay flow cytometry antibody recommended
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