Cd47 fitc

PBMCs were extracted from healthy subjects. PBMC samples were divided into the Control, PBC, IFN-α, and TNF-α groups. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). According to the grouping, 100 uL of healthy human serum, 100 uL of PBC patient serum, IFN-α (#ab200262, absin, China) (0/10/50) ng/mL, or TNF-α (#ab259410, absin) (0/10/50) ng/mL was added to PBMC (10⁵ cells/mL), mixed and incubated at 37°C, 5% CO₂ incubator overnight (12 h). Cells were collected and treated with Fixable Viability Dye eF780(#65-0865, eBioscience, USA) and FC receptor blockers (#564765, BD Biosciences, Germany). After washing, 5 μL of fluorescent dye CD14-BV510 (#563079, BD Biosciences) and 20ul of CD47-FITC (#556045, BD Biosciences) were added and incubated at 4°C in the dark for 30 min. After washing, the cells were detected by CytoFlex V5-B5-R3 Flow cytometer (No: 38385, Beckman USA) Flow cytometer.

After treated with Fixable Viability Dye eF780(#65-0865, eBioscience, USA) and FC receptor blockers (#564765, BD Biosciences, Germany), PBMCs (10⁵ cells/mL) were incubated for 30 min at 4°C in the dark with 5ul of fluorescent dyes CD3-Percp-Cy5.5 (#560835, BD Biosciences), CD19-BV421 (#562440, BD Biosciences), CD14-BV510 (#563079, BD Biosciences), CD56-BV650(#564057, BD Biosciences), SIRPα-APC (#17-1729-42, eBioscience), 20ul of CD16-PE (#555407, BD Biosciences) or CD47-FITC (#556045, BD Biosciences). After washing, the cells were detected by CytoFlex V5-B5-R3 Flow cytometer (No: 38385, Beckman USA). The expressions of CD47 and SIRPα in CD3⁺ T cells, CD19⁺ B cells, CD56⁺ NK cells or CD14⁺ monocytes in PBMC were analysed using flowjo 6.2 software. The gate-drawing strategies for CD3⁺ T cells, CD19⁺ B cells, CD56⁺ NK cells and CD14⁺ monocytes were shown in Figure 1.