Cultured dendritic cells from mouse bone marrow precursor cells
Cultured dendritic cells from mouse bone marrow precursor cells
Dendritic cells (DCs) are very few in number in vivo (less than 1% of splenic adherent cells), and the time-consuming isolation of DCs from vivo and their low cell yields have greatly slowed down the study of DCs. Therefore, it is important to establish a method to culture a large number of DCs from precursor cells in order to study the biological functions of DCs.
Principle
The basic principle of mouse bone marrow precursor cell culture of dendritic cells is that mouse bone marrow contains a large number of hematopoietic stem cells, and the precursor cells of DC can be induced to differentiate and develop into DC by GM-CSF and IL-4. This method is the most commonly used method for obtaining dendritic cells in mice, as it allows us to obtain a sufficient number of dendritic cells with the same differentiation and developmental status for research purposes. It should be noted that although the naturally occurring mouse DCs consist of three subpopulations, CD4+CD8, CD4CD8+ and CD4-CD8-, in vitro culture can only produce the CD4-CD8 subpopulation; moreover, different laboratories have different degrees of improvement in the process of applying this method according to their own experience.
Operation method
Cultured dendritic cells from mouse bone marrow precursor cells
Principle
The basic principle of mouse bone marrow precursor cell culture of dendritic cells is that mouse bone marrow contains a large number of hematopoietic stem cells, and the precursor cells of DC can be induced to differentiate and develop into DC by GM-CSF and IL-4. This method is the most commonly used method for obtaining dendritic cells in mice, as it allows us to obtain a sufficient number of dendritic cells with the same differentiation and developmental status for research purposes. It should be noted that although the naturally occurring mouse DCs consist of three subpopulations, CD4+CD8, CD4CD8+ and CD4-CD8-, in vitro culture can only produce the CD4-CD8 subpopulation; moreover, different laboratories have different degrees of improvement in the process of applying this method according to their own experience.
Materials and Instruments
Reagents: Move The basic process of mouse bone marrow precursor cell culture of dendritic cells can be divided into the following steps: (1) Aseptically take mouse femur and tibia and place them in RPMI 1640 medium at 4 ℃. After poking through the epiphysis with a 1 mL syringe, aspirate the RPMI 1640 medium several times and rinse the bone marrow cavity repeatedly until the bone turns white in order to obtain enough bone marrow cells. (2) Filter the obtained bone marrow cells through a sieve (to remove particles) and collect them in a centrifuge tube, centrifuge at 280 g for 5 minutes at room temperature, and discard the supernatant. (3) Add 3-10 mL of erythrocyte lysate to lysed erythrocytes. After 3 minutes at room temperature, centrifuge at 280 g for 5 minutes at room temperature and discard the supernatant. (4) Wash bone marrow cells twice with RPMI 1640 and centrifuge at 280 g for 10 minutes at room temperature. Count the viable cells and adjust the cell concentration to 1x106 cells/mL to 2x106 cells/mL using complete RPMI 1640 medium. (5) Add mouse recombinant GM-CSF to a final concentration of 10-20 ng/mL and mouse recombinant IL-4 to a final concentration of 1 ng/mL. Inoculate 4 mL of cell suspension per well into a 6-well plate. (6) After 3 days, colonies can be seen growing on the bottom of the plate. Gently shake the plate to suspend the non-adherent cells in the liquid, tilt the plate, remove the medium, gently add RPMI 1640 to the wells along the walls, gently shake the plate and then aspirate the washing solution, and finally add 4 mL of complete RPMI 1640 medium containing 10-20 ng/mL rmGM-CSF and 1 ng/mL rmIL-4 to each well. (7) Between day 5 and day 8 (when enough aggregates have been produced), half-volume changes were made every 1 to 2 days (tilt the plate, remove 2 mL of medium, and then add 2 mL of complete RPMI 1640 medium containing 10-20 ng/mL of rmGM-CSF and 1 ng/mL of rmIL-4 along the wall of each well. (8) After day 8, detached cells were collected and centrifuged at 280 g for 10 minutes at room temperature, and the supernatant was discarded. The cells were resuspended in complete RPMI 1640 medium containing 10-20 ng/mL rmGM-CSF and 1 ng/mL rmIL-4 up to a maximum concentration of 1x106 cells/mL, and then spread in 100 mm dishes at 10 mL per dish. (9) Gently shake the dishes every 24 hours for 24 to 48 hours after cell transfer to collect non-adherent, non-proliferating, mature DCs, and transfer them to another collection tube for later study. Caveat (1) The best mice to use are 5-6 week old males with thicker bones and more DC induced; rest for at least 5 days after transportation; do not use stressed or dehydrated mice.(2) The amount and duration of action of ammonium chloride used for lysing erythrocytes is determined empirically.(3) The duration of DC collection depends on the needs of the experiment. For more product details, please visit Aladdin Scientific website.
5-6 weeks old mice, RPMI 1640 medium at room temperature and 4℃, erythrocyte lysate 0.02mol/L Tris-HCl (pH7.2); 0.14mol/L NH4Cl, mouse GM-CSF and IL-4, antibody for DC detection by flow cytometry.
Instruments:
Anatomical surgical equipment, 100 mm Petri dishes, 6-well Petri dishes, 1 mL syringes, 15 mL and 50 mL polypropylene cone-bottomed centrifuge tubes