Cx3cr1 percp cy5

Cryopreserved peripheral blood mononuclear cells (PBMCs) from COBRA participants and BBD were used for immune phenotyping. The PBMCs were thawed and subsequently stained with monoclonal antibodies (mAbs) for 30 minutes at 4°C in the dark, to determine expression of different surface molecules. The following directly conjugated mAbs were used for cell surface marker staining: CD14 PE-Cy7, CD16 eFluor 450, CD32 PerCP-eFluor 710, and CD11c APC (eBioscience, San Diego, CA); CD163 AlexaFluor 488 and CD86 PerCP (R&D Systems, Minneapolis, MN); CX3CR1 PerCP-Cy5.5 (BioLegend, San Diego, CA); HLA-DR V500, CD3 V500, CD4 PE-Cy7, HLA-DR fluorescein isothiocyanate (FITC), and CD38 PE (BD Biosiences, San Jose, CA); CD38 PE and CD91 PE (BD); and CD40 APC-H7, CD64 APC-H7, and CD8 Pacific Blue (BD Pharmingen, San Diego, CA). Fluorescence was measured with the FACS Canto II (BD Biosciences). The proportion of cells and the mean fluorescence intensity (MFI) of the markers was determined using FlowJo 7.6 (TreeStar, Ashland, OR).

Approximately 5.0 × 10⁵ cells were pelleted and resuspended in 100 μL staining buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA). Cells were stained with a 50 μL cocktail containing staining buffer, HLA‐DR‐PE‐Cy7, CCR2‐AmCyan, CX3CR1‐PerCp‐Cy5.5, TLR‐4‐PE, and TLR‐2‐FITC (Biolegend) for 30 mins at room temperature, and washed twice in staining buffer. Cells were resuspended in staining buffer and analyzed using a Beckman Coulter Gallios flow cytometer. Fluorescence is presented as the natural log mean fluorescent intensity (MFI).

As part of the original A5314 study, cryopreserved PBMC specimens from A5314 (n=118) were thawed and stained with LiveDead Yellow (ThermoFisher), CD3-BV711 (clone UCHT1, BD), CD4-AlexaFluor(AF)-700 (RPA-T4, BD), CD8-BrilliantViolet(BV)-785 (RPA-T8, BD), HLA-DR-FITC (L243, BD), CD38-APC (HIT2, BD), CD14-Pacific Blue (M5E2, BD), CD16-APC-Cy7 (3G8, BioLegend), CX3CR1-PerCP-Cy5.5 (2A9-1, BioLegend), and CD69-PE-Cy7 (L78, BD). Additional aliquots of cryopreserved PBMCs from each donor at baseline and week 24 were then thawed and stained with a second panel, consisting of LiveDead Aqua (ThermoFisher), CD3-BV605 (SK7, BD), CD4-BrilliantUltraViolet(BUV)395 (SK3, BD), CD8-APC-Cy7 (SK1, BioLegend), CD45RA-APC (HI100, BD), CD27-BV786 (O323, BioLegend), CD95-PE (DX2, BD), CD127-PECF594 (A0195D5, BioLegend), CD39-PerCPCy5.5 (eBioA1, eBioscience), CD73-BV421 (AD2, BioLegend), Ki67-PECy7 (B56, BD), and Bcl-2-FITC (Bcl-2/100, BD). All researchers remained blinded to the treatment status of the donors during flow cytometry acquisition and gating.