Alexa fluor 647 conjugated anti cd4

For detection of Tfh cells, human PBMCs were stained with Alexa Fluor 647-conjugated anti-CD4, Alexa Fluor 488-conjugated anti-CXCR5, and phycoerythrin (PE)-conjugated anti-PD-1 (all from BD Pharmingen, San Jose, CA). Cells were gated for CD4⁺ T cells first and then for CXCR5⁺PD-1⁺ Tfh cells. For detection of Breg cells, PBMCs were stained with PerCP/Cy5.5-conjugated anti-CD19, fluorescein isothiocyanate (FITC)-conjugated anti-CD5, and PE-conjugated anti-CD1d (eBioscience) for 15 minutes. CD5⁺CD1d^high cells were analyzed with a CD19⁺ gate.
For intracellular IL-10 staining, PBMCs were incubated for 24 hours with 10 µg/ml LPS and stimulated with PIB for the last 5 hours. Surface staining with PerCP/Cy5.5-conjugated CD19 or FITC-conjugated anti-CD5 was first performed for 15 min, and cells were re-suspended in Fixation/Permeabilization solution (Invitrogen). Intracellular staining of PE-conjugated anti-IL-10 was performed according to the manufacturer’s protocol (eBioscience). After staining, IL-10⁺ cells were analyzed with a CD19⁺ gate by flow cytometry. For some experiments, cells were stained with FITC-conjugated CD19 and PE-conjugated anti-IL-10 (eBioscience) and detected by immunofluorescence microscopy.

A MACSQuant Flow Cytometer (Miltenyi Biotec) was used to analyze whether the cells utilized the following specific antibodies: Violet Fluor^™ 450-conjugated anti-B220 (Tonbo Biosciences); Alexa Fluor 647-conjugated anti-CD4 (BD Pharmingen); Brilliant Violet 510^™-conjugated anti-CD4 (BioLegend); Brilliant Violet 510^™-conjugated anti-CD8a (BioLegend); Alexa Fluor 647-conjugated anti-GL7 (BioLegend), phycoerythrin (PE)-conjugated anti-CD62L (BD Biosciences); allophycocyanin (APC)-conjugated anti-CD44 (eBioscience); APC-conjugated anti-CD86 (Tonbo Biosciences); PE-conjugated anti-CD69 (BioLegend); PE-conjugated anti-PD-1 (Tonbo Biosciences); Brilliant Violet 421^™-conjugated anti-C-X-C chemokine receptor type 5 (BioLegend); Alexa Fluor 647-conjugated anti-inducible T cell co-stimulator (BioLegend); eFour450-conjugated anti-CD127 (eBioscience); PE-conjugated anti-CD25 (Tonbo Biosciences); and APC-conjugated anti-CTLA4 (Tonbo Biosciences). In addition, dead cells were excluded by propidium iodide staining. FlowJo (FlowJo, LLC) was used to conduct data analysis.