2.3 Flow cytometric analysis
To analyze the proportion of CD56+NK cells and CD161 on the surface of
CD56+NK cells in pSS patients and HCs peripheral blood, we used the
following human fluorescent antibody markers: anti-CD56-FITC (Biolegend,
USA) and anti-CD161-PE(Biolegend, USA). The isotype control antibodies
used in our study were as follows: Mouse IgG 1κ-FITC (Biolegend,
USA),Mouse IgG1κ-PE (Biolegend, USA).
Fresh venous blood of pSS patients and HCs on an empty stomach were
collected with heparinized anticoagulation tubes. According to the
instructions of the manufacturer, 50μL whole blood samples were stained
with human CD56-FITC antibody and human CD161-PE antibody, and incubated
at 4℃in the dark for 30 minutes.Then, 200μL/test of erythrolysin(1×) was
added into all sample tubes, and all samples vibrated by an oscillator
and placed in a thermostat at 37℃ to fully lyse red blood cells. Then,
2mL/test sheath fluid was added into all samples for washing and
centrifugation(1200rpm,5min), and the waste liquid was carefully dumped
(Took and put the test tubes gently to avoid vibration!).
Next,0.5mL/test of sheath fluid was added to resuspend white blood cells
to obtain leukocyte suspension for detection. The flow cytometric
analysis was processed by flow cytometry (FC500, Beckman Coulter, USA),
and the data results were analyzed by FlowJo 7.6 software.