Bv510 conjugated anti cd3

Venous blood samples were collected in EDTA tubes from patients with active MPO-AAV and HC. To obtain the best detection results and minimize cell manipulation, our peripheral blood samples were processed within 4 h and immediately analyzed by FACS. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood on Ficoll Paque-PLUS (GE Healthcare, Little Chalfont, UK) by density gradient centrifugation. Freshly collected PBMCs were stained immediately using the following fluorochrome-conjugated anti-human antibodies: BV510-conjugated anti-CD3, BB515-conjugated anti-CD4, APC-Cyanine7 (APC-Cy7)-conjugated anti-CD8, PerCP/Cyanine5.5-conjugated anti-CD45RO, PE/Cyanine7 (PE-Cy7)-conjugated anti-CCR7, PE-conjugated anti-CCR6, Brilliant Violet 421-conjugated anti-CCR4, and APC-conjugated anti-CXCR3 (BD Biosciences). Briefly, appropriate concentrations of fluorochrome-conjugated monoclonal antibodies specific for cell surface antigens were added to tubes containing 100 μL of PBMCs and incubated for 30 min in the dark at 4°C. Subsequently, cells were washed twice with phosphate-buffered saline/0.01% bovine serum albumin and immediately analyzed using a BD FACS Canto II flow cytometer.

Cells were stained with antibodies for flow cytometry, including Fixable Viability Stain 780(FVS780), BV510-conjugated anti-CD3(UCHT1), APC R700 anti-mTCR(H57-597), BV421 anti-CD137(4B4-1), APC anti-CD69(FN50) (BD Biosciences, Franklin Lakes, NJ, USA). For mTCR and other membrane molecules detection, cell staining steps were performed at room temperature for 15 min with 1 × PBS washes between steps.