Due to the lack of characteristic surface markers of macrophages or macrophages at different stages of differentiation, macrophages are currently identified based on the surface molecules of different types of giant sun cells . Expression profiles of different types of macrophages are combined with various fluorescein-labeled monoclonal antibodies, and macrophages are identified by flow cytometry Author: J.E. Colligan et al.
Operation method
Isolation of mouse macrophages Move Basic protocol direct labeling for the detection of macrophage cell phenotypes in mice Materials Sorvall RT 6000B Centrifuge 2. Add 4ul of 3 mg/ml blocking IgG to each tube and incubate at 4°C for lOmin. 3 . Add appropriate amount of biotin-coupled monoclonal antibody to tubes 1 and 2 as shown in Table 6.2.2 and incubate tubes 1, 2 and 3 on ice for 15 min. 4 . Add 5 M1 EMA to tube 4 and mix well. Place the tube 18 cm away from an incandescent lamp (e.g., 40 W incandescent lamp) at room temperature for lOmin. EMA enters only the dead cells. The strong coloration of dead cells is used to distinguish them from live cells on the FLL and FL3 two-variable scatter plots. 5 . Add 3 m l of PBS to each tube and centrifuge at 1500 g for 3 min. Pour off the supernatant quickly and, with the tube standing upright, flick the bottom of the tube with your finger to loosen the cell deposits. Add F IT C-labeled streptavidin to tube 1 and T C-labeled streptavidin to tube 2. The four tubes are then placed in an ice bath for lOmin, in which the cells in tubes 3 and 4 are loosened without the need for additional liquid. 6 . For samples containing erythrocytes (e.g., freshly isolated bone marrow cells or splenocytes), add 3 ml of ACK lysate to each tube; for samples not containing erythrocytes, add 3 ml of PBS to each tube. 7 . Cover the tubes tightly, turn the tubes upside down 1~2 times, mix the cells well, and let them stand for 3 min at room temperature. 8 . At room temperature, centrifuge at 1500& for 4 min, pour off the supernatant quickly, and when the tube is upright, flick the bottom of the tube to loosen the cell precipitate. 9 . Wash the cells with 3 ml of PBS as described in steps 7 and 8. 10a For direct detection: add 20()/^?83, mix the cells and leave at 4°C under light (can be stored for <4h). For IOb post-fixation assay: add IOOpJ 2% formaldehyde to each tube, mix well, cover, protect from light at 4°C, and fix for Ih post-fixation. Formaldehyde alters the light scattering value of cells. II. Detect by flow cytometry and analyze the results by FACSCAN (Chapter IV). 4 . Add 4ul of normal mouse serum IgG at a concentration of 3m g/m l to each tube. ice bath for lOmin to block the non-specific binding site on the secondary antibody. 5 . Add appropriate amount of fluorescein-labeled anti-F (ab)[fragment secondary antibody to each tube. Mix and ice bath for 15 min. 6 . Wash the cells according to step 9 of the basic protocol. 7 . Optional: Without washing the cells, add an appropriate amount of the second fluorescein-labeled secondary antibody to the tubes to be tested and to tube A. Add an equal amount of fluorescein-labeled mouse IgG to tube B to detect the blocking effect of step 6. An equal amount of fluorescein-labeled mouse IgG is added to tube B to detect the blocking effect of step 6. 8 . For the remaining steps, refer to steps 9 to 11 of the basic protocol. For more product details, please visit Aladdin Scientific website.
I. Prepare four 12m m X 75m m tubes, numbered 1 to 4. Prepare four 12m m X 75m m test tubes by numbering the tubes 1 to 4. Add 2 X IO7 cells/ml of cell suspension 50ul to each tube.