Tritest cd4 fitc cd8 pe cd3 percp reagent

For surface marker analyses, a sample of 50 μL of well-mixed EDTA anticoagulated whole blood was incubated for 30 min at room temperature with 20 μL of BD Tritest CD4 FITC/CD8 PE/CD3 PerCP Reagent (BD Biosciences, San Jose, CA, USA) according to manufacturer instructions. Following incubation, red blood cells were lysed with NaHCO₃/ClNH₄ solution. Cells were washed twice with PBS and preserved for cytometric analysis.
Another sample of 1 × 10⁶ PBMC was incubated for 30 min with anti-CD4-FITC and anti-CD25-PEcy5.5 (BD Biosciences, San Jose, CA, USA) and for intracellular staining of FoxP3, the cells in the tubes were resuspended in Fix/Perm buffer (eBioscience, San Diego, CA, USA) and left at 4°C for 30 min. Subsequently, these cells were washed with cold PBS and with permeabilization buffer (eBioscience, San Diego, CA, USA) and then 20 μL anti-human FoxP3-PE (BD Biosciences, San Jose, CA,USA) was added and the cells were incubated at 4°C for 30 min. Stained cells were analyzed with a FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA). The percentage of positive cells and the mean fluorescence intensity (arbitrary units) for a specific marker were calculated using FACSDiva software (BD Biosciences, San Jose, CA, USA). For purpose analysis 30000 events were recorded.

A sample of EDTA anticoagulated whole blood was incubated with BD Tritest CD4FITC/CD8PE/CD3PerCP reagent, another with CD3FITC/CD19PE, and the third one with CD45FITC/CD14PE (all reagents of BD Biosciences) and isotype controls, according to the manufacturer’s instructions (Díaz et al., 2015 (link)). Stained cells were analyzed with a FACSAria II flow cytometer (BD Biosciences). The percentage of positive cells and the mean fluorescence intensity (arbitrary units) for a specific marker were calculated using FACSDiva software (BD Biosciences). For each sample, 30.000 events were recorded.