Hla dr allophycocyanin cy7

Whole blood and mononuclear cells isolated from intestinal biopsies were stained for flow cytometry using a six-color technique as described previously to assess changes in the levels of major T cell populations and their immune activation status. The mAb combination used was: CD3-Pacific Blue, CD4-allophycocyanin, CD8-Texas Red, HLA-DR-allophycocyanin-Cy7, CD38-PE (BD Biosciences) and Ki-67–FITC (BD Pharmingen). All Abs were validated and titrated using PBMCs from PTMs [17 (link),75 (link)]. Samples were stained for Ki-67 using the Ki-67/FITC–conjugated mouse anti–human mAb set (BD Pharmingen) as per the manufacturer’s instructions. Stained cells were analyzed with an LSRII flow cytometer (BD Biosciences) and FlowJo Version 7.6 software (TreeStar). CD4⁺ and CD8⁺ T cell percentages were obtained by first gating on lymphocytes, then on CD3⁺ T cells. Activation markers were determined by gating on lymphocytes, then on CD3⁺ T cells, and finally on CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells.

Apoptosis was detected using Annexin V apoptosis detection kit FITC (e-Bioscience; catalog number 88-8005-72) per manufacturer’s instructions. Briefly, after harvesting THP-1 and PBMC at the indicated times, cells were centrifuged and washed once with cold PBS followed by another wash with 1X cold Annexin V Binding Buffer. To the cells (10⁶ cells/100μl), 5μL of fluorochrome-conjugated Annexin V was added and incubated in the dark for 15 min. Cells were washed once with 1X Annexin V Binding Buffer and re-suspended in 300μL of 1X Annexin V Binding Buffer containing 5μL Propidium iodide (eBioscience). Percent apoptosis in PBMC subsets (T cells, B cells, dendritic cells, and monocytes) was determined by staining with antibodies (BD Bioscience) specific for immune cell surface markers: HLA-DR-allophycocyanin-Cy7 (Catalog number 335796), CDllc-APC (Catalog number 340544), CD19-PerCP (Catalog number 340421), CD14-Alexa Fluor 700 (Catalog number 557923), CD3-PE-Cy7 (Catalog number 563423). Samples were analyzed using an LSRII flow cytometer (BD Bioscience).