This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Expression of HLA-B27 in peripheral blood leukocytes

Materials and Instruments

CD3 Monoclonal antibody HLA-B27 Monoclonal antibody Whole blood
Hemolytic Agents Paraformaldehyde
Flow Cytometry

Move

1. Take 30 μl of fluorescein PE-labeled CD3 monoclonal antibody and FlTC-labeled HLA-B27 monoclonal antibody, and 50 μl of _{EDTAK3-anticoagulated} whole blood, and then mix well and stain for 15 min (or 20 min) at room temperature.

2. Add 2 ml of hemolytic agent to dissolve the red blood cells (10 min).

3. Remove cell debris by centrifugation at 1500 r/min, and fix with 1% paraformaldehyde (15 min).

4. Measurement on the machine. After measuring the standard microspheres and adjusting the Fl1 voltage, do not change the FL1 setting. Set the "gate" by CD3-PE/SSC to circle the CD3-positive cells (T lymphocytes), and detect the average fluorescence intensity of their B27. A specimen is negative if its fluorescence intensity is less than the standard microsphere fluorescence intensity, and positive if the opposite is true.

5. Give the flow cytometry results and issue a lab report.

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