Induction and isolation of human blood mononuclear macrophages
Induction and isolation of human blood mononuclear macrophages
Macrophages (MΦ) are mature cells formed by the migration of blood monocytes into tissues, and are widely distributed in the spleen, liver, abdominal cavity, nervous system, and connective tissues, with phagocytosis and killing, antigen presentation, and immunomodulatory effects. Recent studies have found that macrophages can be activated in different microenvironments and become subpopulations with different surface molecular and functional characteristics. According to their different phenotypic and functional characteristics and induction of Th1/Th2 responses, they are usually classified into two types: classically activated macrophages (CAM), also known as M1-type macrophages; alternatively activated macrophages (AAM), also known as M1-type macrophages; and alternatively activated macrophages (AAM), also known as M1-type macrophages. M1 macrophages are microbicidal, inflammatory, and antitumor, while M2 macrophages have strong immunomodulatory, tissue repair, and anti-inflammatory properties.
Principle
The basic principle of human blood monocyte macrophage Ficoll density gradient centrifugation is that the specific gravity of the layered liquid sucrose (Ficoll)-Urografin is 1.077 ± 0.001. The density of Ficoll is 1.2 g/ml, which is not beyond the normal physiological osmolality, and it does not cross the biofilm, therefore, it does not affect the morphology and function of the single nucleated cells. Erythrocytes and granulocytes have a high specific gravity (1.092) and sink to the bottom of the tube after centrifugation; lymphocytes and monocytes have a specific gravity less than or equal to the specific gravity of the stratified liquid (1.070) and float on the surface of the stratified liquid after centrifugation, and the cells that are sucked up from the liquid surface of the stratified liquid are peripheral blood mononuclear cells. Based on the characteristics of monocyte macrophage that can be attached to the wall, the walled monocyte macrophage is further separated from the lymphocyte that is not attached to the wall. When monocytes are cultured in vitro, a certain amount of rhGM-CSF can be added to promote the transformation of monocytes into macrophages.
Operation method
Separation of human hemorrhagic mononuclear macrophages by Ficoll density gradient centrifugation
Principle
The basic principle of human blood monocyte macrophage Ficoll density gradient centrifugation is that the specific gravity of the layered liquid sucrose (Ficoll)-Urografin is 1.077±0.001. The density of Ficoll is 1.2 g/ml, which is not beyond the normal physiological osmolarity, and it does not cross the biofilm, and thus does not affect the morphology and function of single nucleated cells. Erythrocytes and granulocytes have a high specific gravity (1.092) and sink to the bottom of the tube after centrifugation; lymphocytes and monocytes have a specific gravity less than or equal to the specific gravity of the stratified liquid (1.070) and float on the surface of the liquid after centrifugation, and the cells that are sucked up on the surface of the liquid of the stratified liquid are the peripheral blood mononuclear cells. Based on the characteristics of monocyte macrophage that can be attached to the wall, the walled monocyte macrophage is further separated from the lymphocyte that is not attached to the wall. When monocytes are cultured in vitro, a certain amount of rhGM-CSF can be added to promote the transformation of monocytes into macrophages.
Materials and Instruments
Sample: Move The basic procedure for induction and isolation of human blood mononuclear macrophages can be divided into the following steps: Caveat (1) When adding human peripheral blood to the lymphocyte separation solution, you should move gently and do not break the separation interface, otherwise the separation effect will be affected.(2) When aspirating the monocyte layer, try to gently aspirate all of them to minimize the mixing of cells in other layers.(3) Strict attention should be paid to aseptic operation when changing fluids. For more product details, please visit Aladdin Scientific website.
Human anticoagulated peripheral blood
Reagents:
Lymphocyte isolate, RPMI 1640 with 10% calf serum (penicillin 20,000 IU/ml streptomycin 200,000 IU/ml), rhGM-CSF, 3% acetic acid, PBS, anti-CD14, anti-CD68, or anti-F4/80 labeled antibodies.
Instruments:
Centrifuge tubes, pipettes and tips, EP tubes, centrifuge, light microscope, cell counting plates, rubber rods, coverslips, alcohol wipes, CO2 incubator, ultra-clean bench, 6-well plate, flow cytometer or magnetic bead sorter.
(1) Firstly, 5 ml of lymphocyte layering solution was added to the centrifuge tube, and then 5 ml of heparin-treated anticoagulated human venous blood was gently added and centrifuged horizontally (2,000 r/min for 15 minutes).
(2) After centrifugation, use a capillary tube to collect a narrow band of white nebulosity (i.e., the layer of single nucleated cells) at the interface of the upper and middle layers into another centrifuge tube. Add 5 times the volume of PBS, mix well and centrifuge (1500 r/min, 15 min), discard the supernatant. Repeat twice and discard the supernatant.
(3) Flick the bottom of the tube to disperse the cells, then add 2 ml of RPMI1640 medium containing 10% calf serum and resuspend the cells.
(4) Count the cells, adjust the cell concentration to 5 x 106/ml, add them to a 6-well plate, 2 ml/well, and incubate at 37 ℃ in 5% CO2 incubator for 2-4 hours, then gently wash the cells with PBS for 3 times to wash away the unadhered cells.
(5) Add 2 ml of RPMI 1640 medium containing 10% fetal bovine serum and rhGM-CSF (final concentration 1000 IU/ml) to each well, incubate at 37 ℃ with 5% CO2 for 3-5 days, and then change the solution in half every 2 days.
(6) Rinse the culture plate with PBS and repeat 3 times to discard the suspended cells. Then, add appropriate amount of culture medium and collect them by gently scraping with a rubber mallet to obtain wall-adherent macrophages and count them.
(7) For further purification, surface marker CD14 or F4/80 can be used for labeling, and the specific steps are the same as flow cytometry separation and immunomagnetic bead method.