Anti mhc class 2 pe

Cells (2 × 10⁵ per well) were blocked on ice using 1 μL/mL Serobloc in blocking buffer (phosphate‐buffered saline [PBS], 5% heat inactivated normal rabbit serum (HI‐NRS), 1% bovine serum albumin (BSA), 2 mM sodium azide), and stained with anti‐CD11c‐APC (BD Pharmingen, Wokingham, Berks, UK; Cat# 550261), anti‐CD40‐FITC (BD Pharmingen, Cat# 553790), anti‐CD54‐PE (BD Pharmingen, Cat# 553253), anti‐CD80‐PE (BD Pharmingen, Cat# 553769), anti‐CD86‐PE (eBioscience, San Diego, California, Cat# 12‐0861‐81), anti‐H‐2K^k‐PE (BD Pharmingen, Cat# 553593), anti‐MHC class II‐PE (eBioscience, Cat# 12‐5321‐81) or anti‐SSEA‐1‐PE (eBioscience, Cat# 12‐8813‐71). For analysis of transcription factor expression, cells were permeabilized with 0.1% saponin for 5 minutes and stained using anti‐Nanog‐Alexa Fluor 488 (BD Pharmingen, Cat# 560278) or anti‐Oct4‐Alexa Fluor 647 (BD Pharmingen, Cat# 560329). Samples were washed three times in washing buffer (PBS, 1% FCS, 2 mM NaN₃), fixed in 2% formaldehyde/PBS, and analyzed using a FACSCalibur cytometer.

The maturation of DCs was examined by staining with fluorescence‐conjugated antibodies including anti‐CD11c‐FITC (eBioscience, catalog: 11‐0114‐85), anti‐CD86‐APC (eBioscience, catalog: 17‐0862‐82), and anti‐MHC Class II‐PE (eBioscience, catalog: 12‐5321‐82). To analyze the T‐cell subsets in spleens and draining lymph nodes of the immunized mice, single cell suspensions prepared from these samples were examined by flow cytometry. The following primary antibodies were used: anti‐CD3e‐PerCP‐Cyanine5.5 (eBioscience, catalog: 45‐0031‐82), anti‐CD8a‐PE (eBioscience, catalog: 12‐0081‐82), anti‐CD4‐FITC (eBioscience, catalog: 11‐0041‐85), anti‐CD25‐APC (eBioscience, catalog: 17‐0251‐82), anti‐Foxp3‐PE (eBioscience, catalog: 12‐4771‐82), anti‐IFN‐γ‐FITC (BD Bioscience, catalog: 554 411), anti‐TNF‐α‐Alexa Fluor 647 (BD Bioscience, catalog: 557 730), and anti‐CD16/CD32 (eBioscience, catalog: MA5‐18012). Flow data were acquired on a CytoFLEX and analyzed using CytExpert (Beckman Coulter, USA) and FlowJo (TreeStar, USA) softwares.