Percp cyanine 5.5 anti human cd4

Cells were collected and washed 2 to 3 times. The cell surface mixed antibody was divided equally into each sample staining with the Zombie aqua fixable viability kit (423101, BioLegend), and continued simultaneously for 30 min at room temperature. After the cell surface staining was finished, the intracellular maker staining was performed. Cell fixation (420801, BioLegend) and staining permeabilization (421002, BioLegend) were required prior to intracellular staining. Flow cytometry determined the expression of distinct surface and intracellular molecules. The following antibodies were used: APC/Cyanine7 anti-human CD3 (300317, BioLegend), PerCP/Cyanine 5.5 anti-human CD4 (317427, BioLegend), PE/cyanine7 anti-human CD8a (300913, BioLegend), Brilliant Violet 421 anti-human IFN-γ (502531, BioLegend), and PE/DazzleTM594 anti-human CD107a (LAMP-1) (328645, BioLegend) and APC anti-human TNF-α (502913, BioLegend), APC anti-human CD19 (302211, BioLegend). The apoptosis of BJAB cells was assayed using the FITC Annexin V Apoptosis Detection Kit with PI (640914, BioLegend). The data were analyzedusing FlowJo 10.8 software (https://www.flowjo.com/).

The following monoclonal antibodies (Abs) were used for surface phenotype and intracellular cytokine staining: peridinin chlorophyll protein (PerCP)/Cyanine5.5 anti-human CD4, fluorescein isothiocyanate anti-human CD45RA, Alexa Fluor 700 anti-human CD183 (CXCR3), Brilliant Violet 510 anti-human CD196 (CCR6), allophycocyanin (APC) anti-human CCR10 (all from Biolegend), Brilliant Violet 421 anti-human CD25, phycoerythrin (PE)-CF594 mouse anti-human CD194 (CCR4) (all from BD Biosciences), and PE anti-human Foxp3 (from eBioscience). Precision Count Beads (from Biolegend) were used to obtain absolute cell counts. Cells were acquired and analyzed using the CytoFLEX S flow cytometer (Beckman) and CytExpert software.