Hla b8 pe

HLA-ABC and HLA-B8 expression levels in cell lines were analyzed by staining with HLA-ABC-FITC (BD) and HLA-B8-PE (Miltenyi Biotec) antibodies, respectively. The HLA-negative cell line K562 was used to set the gates for HLA-ABC⁺ and HLA-B8⁺ cells. Data were acquired using a BD Canto II flow cytometer and analyzed using FlowJo. The antibodies used in this study are listed in online supplemental table S2.

Single-cell suspensions from lamina propria were analyzed by flow cytometry according to standard procedure using fluorochrome-conjugated antibodies targeting HLA-A2–PE (Abcam); CD20-FITC, CD45-APC-H7, and Ki67-FITC (BD); Bcl2–Alexa Fluor 488, BCMA-PE, CD3-APC, CD14-APC, CD19–Alexa Flour 488/PE-Cy7, CD20-PE, CD27-BV421/Pacific blue/APC-Cy7/PE-Cy7, CD28–Alexa Fluor 488, CD38-PE/APC-Cy7, CD45-BV510/APC-Cy7, CD56–Alexa Flour 488, CD95-PE, and HLA-DR–BV605/PerCP (BioLegend); CD138-FITC/PE-Cy7 and HLA-A3–FITC/APC (eBioscience); HLA-B7–PE (EMD Millipore); HLA-B8–PE (Miltenyi Biotec); IgA-FITC/PE, IgM-FITC/PE, and IgD-FITC/PE (SouthernBiotech); and Blimp-1–DyLight 488 (Thermo Fisher Scientific). For detection of intracellular antigens, cells were fixed in formaldehyde and permeabilized using a Foxp3 staining buffer set (eBioscience). Detection of rotavirus-specific PCs was performed using eGFP-tagged virus-like particle 2 (VLP2)–eGFP/VLP6 (provided by D. Poncet and A. Charpilienne, Institut de Biologie Integrative de la Cellule, Paris, France; Charpilienne et al., 2001 (link)) at a final concentration of 1 µg/ml. eGFP (Promega) was used as a negative control. All flow cytometry was performed on an LSRFortessa, and FACS was performed on a FACSAriaII flow cytometer (both from BD). Data were analyzed using FlowJo 10 (Tree Star), and figures were assembled in Illustrator CS4 (Adobe).