Preparing samples for flow cytometry

　　1.1 Preparation of adherent cell samples

　　1.1.1 Aspirate the cell supernatant and wash it twice with PBS. Add a small amount of 10 mM EDTA solution (cover the bottom of the cell culture dish) to gently digest the adherent cells. Place it in the incubator for about 5 minutes. Observe under the microscope to see if the cells change. When the cells are round, pipet evenly with 3% FBS-PBS and collect the cells. Centrifuge at 1000 rpm for 5 minutes. After centrifugation, wash once with 3% FBS-PBS. Collect the cell pellet and place it on ice.

　　1.1.2. Prepare primary antibody solution: Dilute according to the dilution ratio in the antibody instruction manual. Incubate every 1×10^6 cells/100μl primary antibody. Incubate on ice for 40 minutes. Mix gently during the incubation process.

　　1.1.3. Wash the cells twice with 1 ml of ice-cold 3% FBS-PBS, centrifuge at 4°C, 1000 rpm for 5 min at low temperature, and collect the precipitate.

　　1.1.4. Prepare secondary antibody solution: dilute according to the dilution ratio in the antibody instruction manual, incubate every 1×10^6 cells/100μl secondary antibody, and incubate on ice in the dark for 30 minutes. Mix gently during the incubation process.

　　1.1.5. Wash cells with ice-cold 3% FBS-PBS, centrifuge at 4°C, 1000 rpm for 5 minutes, collect cell pellets, resuspend each pellet in 300 uL ice-cold 3% FBS-PBS, and then place On ice, protected from light, prepare upflow for sample analysis.

　　1.2 Sample preparation of spleen cells

　　1.1.1 Use sterile surgical scissors to cut the spleen tissue into pieces;

　　1.1.2 Place the cell strainer on a 50 mL centrifuge tube and rinse the strainer with 2 mL of RPMI 1640 complete culture medium;

　　1.1.3 Transfer the supernatant and spleen tissue from the culture dish to the filter, and use the flat end of a sterile syringe to grind the spleen 5 times in a gentle circular motion to release the cells;

　　1.1.4 Rinse the filter with 3 mL of RPMI 1640 complete medium, centrifuge at 400×g for 5 minutes to collect cells, and discard the supernatant;

　　1.1.5 Add 2 mL of red blood cell lysate and mix well, let it stand for 1 minute, centrifuge at 400×g for 5 minutes, and discard the supernatant;

　　1.1.6 Add RPMI 1640 complete medium, resuspend the cells, pass through a cell strainer, and count the cells;

　　After counting, perform experimental operations according to antibody incubation prepared from adherent cell samples.