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    Home > Biochemistry News > Biotechnology News > The spleen cells and tumor cells (MDA-MB-231) were cultured together and the preliminary experiments of tumor immunology research were carried out.

    The spleen cells and tumor cells (MDA-MB-231) were cultured together and the preliminary experiments of tumor immunology research were carried out.

    • Last Update: 2020-09-07
    • Source: Internet
    • Author: User
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    Tumor immunotherapy has shown great potential in the clinical treatment of tumors, making it a late stage in the field of cancer treatment research, and has gradually developed into the fourth largest treatment after surgery, radiotherapy and chemotherapy.
    that the three classic therapies are too old-fashioned and arrogant, but the wave is really strong, and a large number of researchers are boiling over it.
    the current research on tumor immunotherapy around the tumor immune micro-environment at home and abroad, there are many bright spots.
    in the course of tumor immunology research, it is an essential research link to construct tumor immunology research model to verify the effectiveness of tumor immunology research strategy and explore its internal mechanism.
    , the most widely used in-body research model is the co-culture model of immune cells and tumor cells.
    Because the spleen is the body's largest immune organ, containing a large number of lymphocytes and macrophages, is the center of the body's cell immunity and body fluid immunity, so this paper will use spleen cells as an example, detailed how to co-culture spleen cells and tumor cells (MDA-MB-231) and carry out preliminary experiments on tumor immunology research.
    The flowchart of the experiment, as shown in Figure 1, is divided into three parts: First, the extraction and separation of mouse spleen cells cultureD C57BL/6 mice (6 to 8 weeks) cervical dislocated to death, quickly put in 75% ethanol (v/v) soaked for 5 minutes, removed, placed in a sterile petri dish in the ultra-clean table;
    With tweezers clamped the abdominal skin of the mouse, cut a small mouth in the middle of the left abdominal side of the mouse, tear open the skin, that is, visible red long striped spleen; After gently scissoring the spleen with ophthalmology, place it on the 200-purpose cell screen, gently press the spleen with 10 mL syringe needle core, after squeezing, draw a small amount of RPMI1640 medium to rinse the cell screen, rinse the spleen cell suspension into the petri dish;
    10 mL injection needle core using a disposable sterile package syringe; 300 x g centrifugation 5 min at 4 degrees C, go to the top, add 5 mL erythocyte lysate, gently blow evenly with the pipet, lysate at room temperature for 2 minutes (keep shaking gently), add 15 mLPBS Mixing, 300 x g centrifugation 5 min after discarding, washing 2 times with PBS, collecting cells, with RPMI1640 complete culture base (containing 10% FBS, the same below) rehanging cells; Adjust the cell concentration to 5.0 x 106 cells/mL with RPMI1640, spare.
    II, CFDA-SE labeled MDA-MB-231 cells Take 5'L10 mM of CFDA-SE reserve liquid added 5 mL PBS to make 10 m of CFDA-SE solution;
    collected MDA-MB-231 cells digested by trypsin, 200 x g centrifugation 5 min, PBS cleaning cells 3 times, the centrifuged cell group with 1 mL PBS resuscuing and then added 1 mL of 10 mM CFDA-SE solution, incubation at room temperature 8 min and slight concussion; Note: After collecting MDA-MB-231 cells, the serum composition should be cleaned and removed with PBS to avoid affecting the dyeing efficiency of the later CFDA-SE on the cells.
    incubation, add 10 mL of RPMI 1640 full culture, gently blow evenly, to terminate CFDA-SE staining; 200 x g centrifugal 5 min, go to clear, and wash cells 2 times with PBS; rehang and count with RPMI 1640 full culture base; adjust cell concentration to 1 x 105 cells/mL with RPMI 1640 full culture base, spare.
    , spleen cells and CFDA-SE-labeled MDA-MB-231 cells cultured in 12-well plate with 0.5 mL 1 x 105 cells/mL CFDA-SE labeled MDA-MB-231 cells and 0.5 mL 5.0 x 106 cells/mL of spleen cells, shake evenly, put into the cell culture box for a total of 4 hours; Note: Since the spleen cells are semi-suspended cell states, the drug should be given by adding the drug solution directly, and the culture base in the hole should not be absorbed first.
    Incubation, the culture base in the hole is collected into the centrifuge tube, and the walled MDA-MB-231 cells are digested with trypsin, the cells are collected into the centrifugal tube; The PI staining fluid in the apoptosis PI dyeing kit was used for dyeing, and the cell ratio of CFDA-SE/PI plus was the proportion of tumor cells killed after immunomodulating drugs stimulated spleen cells to play an anti-tumor immune effect (Figure 2).
    As can be seen from Figure 2, the apoptosis rate of tumor cells was 9.3% due to spontaneous apoptosis of tumor cells and immune killing of spleen cells without immunomodulation drugs, and after the addition of immunomodulation drugs, the apoptosis rate of tumor cells increased significantly to 30.3%.
    source: Dr. De-Helix Research Assistant.
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