Anti rat igg2a pe

For each patient, we plated all cells extracted from esophageal biopsies in 96-well flat -bottomed plates (NEST Biotechnology, Shanghai, China). Peripheral blood mononuclear cells from normal volunteers were used as the control to generate gating strategy. The plates were centrifuged at 1000 r.p.m. for 3 min at 4 °C, and then the supernatants were discarded. After discarding the supernatant, cells were incubated for 30 min on ice in wash buffer with primary antibodies against anti-human anti-CD3-Vioblue (Mylteny Biotec, Inc., Auburn, CA, USA), anti-CD4-PerCP Cy5.5 (Biolegend, San Diego, CA, USA), anti-CD8-V500 (BD Biosciences), anti-TCRαβ-FITC (eBioscience, San Diego, CA, USA) or anti-CD11b-PE Cy7 (Biolegend) as described previously.^{36 (link)} Control cells were incubated with anti-mouse IgG1-allophycocyanin (eBioscience), anti-mouse IgG1-FITC (Biolegend), anti-rat IgG1-phycoerythrin (PE) (eBioscience), 1:5 dilution of anti-rat IgG2a-PE (eBioscience) or anti-mouse IgG1-PE Cy7 (Biolegend). The cells were then washed in wash buffer and resuspended in 200 μl of wash buffer and transferred into fluorescence-activated cell sorting tubes. Cells were analyzed with flow cytometry on FACS-LSRII (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).