Lymphoid tissue
1、Collect the tissue (spleen, thymus, lymph node) into a tissue culture dish containing 10 mL of flow cytometry staining buffer or other preferred staining buffer. Use a 3-mL syringe to grind the tissue into a single-cell suspension. Alternatively, use 10 mL of flow cytometry staining buffer and grind the tissue between two frosted microscope slides.
2、Place a cell filter on top of a 15 or 50-mL conical tube. Pass the cell suspension from the tissue culture dish through the cell filter to remove cell clumps and debris.
3、Centrifuge the cell suspension at 300-400 x g for 4-5 minutes at 2-8°C. Discard the supernatant.
Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or other recommended antibody staining buffer, then perform cell counting and viability analysis.
4、Centrifuge the cells at 300-400 x g and resuspend them in an appropriate volume of flow cytometry staining buffer or other preferred staining buffer to achieve a final concentration of 1 x 107 cells/mL (other cell concentrations may be applicable for different experiments).
Non-lymphoid tissue
1、Collect the tissue and cut it into 2-4 mm pieces using scissors or a scalpel.
2、Add an appropriate volume of enzyme (dissolved in PBS) according to the enzyme’s instructions and incubate at the optimal temperature for a suitable duration.
3、Gently disperse the cells with a pipette and filter the cell suspension through a cell filter to remove cell clumps and debris. Collect the cell suspension in a conical tube.
4、Centrifuge the cell suspension at 300-400 x g for 4-5 minutes at 2-8°C. Discard the supernatant.
Resuspend the cell pellet in PBS.
5、Centrifuge the cells using the method described in step 4.
6、Repeat steps 5 and 6.
7、Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or other recommended antibody staining buffer, then perform cell counting and viability analysis.
8、Centrifuge the cells using the method described in step 4 and resuspend them in an appropriate volume of flow cytometry staining buffer or other preferred staining buffer to achieve a final concentration of 1 x 107 cells/mL (other cell concentrations may be applicable for different experiments).
Peripheral blood lysis
1、Dilute 10x red blood cell lysis buffer with distilled water to make 1x buffer, and prepare fresh each time.
2、Collect whole blood into an appropriate anticoagulant tube.
3、Pipette 100 μL of mixed anticoagulated blood into a flow tube.
4、Add appropriate amounts of antibodies according to the antibody’s instructions, mix well, and incubate for a suitable duration.
5、Add 2 mL of 1x FCM lysing solution, vortex and mix well.
6、Incubate at room temperature in the dark for 15 minutes.
7、Centrifuge at 300-400 x g for 5 minutes, discard the supernatant. Add 2 mL of PBS, centrifuge at 300-400 x g for 5 minutes.
8、Discard the supernatant and resuspend the cell pellet in 0.5 mL of PBS.
9、Analyze on a flow cytometer within 2 hours.
