2.6 Flow Cytometry Detection of CD3⁺, CD4⁺, and CD8⁺ T Lymphocyte Counts

2.6 Flow Cytometry Detection of CD3⁺, CD4⁺, and CD8⁺ T Lymphocyte Counts

Collect blood from the mice's eyeballs, add it to EDTA-K₂ anticoagulant tubes, mix thoroughly, refrigerate at 4°C for 20 minutes, then take 100 μL of whole blood from each anticoagulant tube, divide into test tubes, and add 5 μL each of Anti-Mouse CD3e PE-cy5 Conjugated, Anti-Mouse CD4 FITC, and Anti-Mouse CD8 PE single-color fluorescent antibodies to each tube. Protect from light, incubate at room temperature for 20 minutes, then add 2 mL of Optilyse C hemolysin, mix well, let stand for 10 minutes, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash twice with PBS, set the excitation wavelength to 488 nm and the emission wavelength to 576 nm, run the machine for detection. Use CELLQuest software to obtain parameters and analyze data: X-axis represents forward scatter, Y-axis represents side scatter, determine the T lymphocyte population, count 10,000 cells within the counting window, mark the results on the histogram using logarithmic scales, adjust the magnification factor, and then measure the positive expression rates of lymphocyte subpopulations.