Anti cd8 pe dazzle594

After cell culture, whole blood samples (10x10⁵ cells/mL) were incubated with the following panel: antibodies from BioLegend, San Diego, CA, USA: anti-CD45-PerCP (Clone: HI30), anti-CD3-AF647 (Clone: UCHT1), anti-CD14-PECy7 (Clone: M5E2), anti-CD4-APC/Cy7 (Clone: OKT4), anti-CD8-PE/Dazzle594 or BV510 (Clone: SK1). After 15 min in the dark, blood was washed once with PBS (1 mL) by centrifugation at 900×g for 5 min at room temperature (RT); then, Fixation buffer was added (100 μL, Cat: 420801, BioLegend, San Diego, CA, USA), and the samples were incubated for 20 min. Then, samples were washed twice with 1 mL of Intracellular Staining Perm Wash buffer (Cat: 421002, BioLegend, San Diego, CA, USA); after the second wash, they were mixed with monoclonal antibodies against cytokines from BioLegend, San Diego, CA, USA: anti-TNFα-BV421 (Clone: MAb11), anti-IL-6-PE (MQ2-13A5), anti-IL-1β-FITC (Clone:JK1B-1), anti-IFNγ-BV421 (Clone:4S. B3), anti-IL-8a-PE (Clone: E8N1). For the exclusion of dead cells, a Zombie Aqua fixable viability kit (BioLegend, San Diego, CA, USA) was added and incubated for 30 min at RT. Last, the mixture was washed once with PBS. At least 30,000 events were acquired in a FACSAria IIu (BD, San Jose CA) flow cytometer. Analysis was performed using Infinicyt^™ Software 1.8.

Cells were surface-stained with anti-CD3-FITC, anti-CD4-AlexaFluor700, anti-CD8-PE/Dazzle594, anti-CD127-PE-Cy7, anti-CCR6-BrilliantViolet421, anti-CCR4-PE, and anti-CXCR3-PerCP/Cy5.5 (Biolegend). Cells were fixed and permeabilized to stain for the intracellular transcription factor FoxP3 using anti-FoxP3-AlexaFluor647 according to manufacturer’s instructions (eBioscience). Cell fluorescence was measured using a LSR II Fortessa flow cytometer (BD Biosciences) and analysis was performed using FlowJo software (Tree Star).