Cd14 pe

Phenotyping of resting and activated moDCs was performed by flow cytometry using anti-human CD1d-phycoerythrin (PE), CD103-FITC, HLA-DQ-FITC, PD-L1-PE (BD Biosciences, Franklin Lakes, NJ, USA), CD1a-allophycocyanin (APC), CD40-FITC (BioLegend, San Diego, CA, USA), CX₃CR1-PE, CD80-FITC, CD83-FITC, CD86-PE, DC-SIGN-FITC, CCR7-PE, CD14-PE (R&D Systems, Minneapolis, MN, USA), B7RP1 (ICOSL)-PE (EBiosciences, Santa Clara, CA, USA), and isotype-matched control antibodies. The ratio of regulatory T-lymphocytes was measured by flow cytometry using anti-human CD25-PE (BD Pharmingen), CD4-FITC (BioLegend), FoxP3-APC (R&D Systems), and anti-IL-10-AlexaFluor488 (BioLegend). The viability of moDCs was determined with 2 μg/ml 7-amino-actinomycin D (LKT Laboratories Inc., St. Paul, MN, USA) dye followed by a 24-h activation period with live bacteria or LPS. Fluorescence intensities were measured by FACSCalibur (BD Biosciences), and data were analyzed by the FlowJo software (Tree Star, Ashland, OR, USA).

Cells were stained with CD3 FITC (Cat# 11-0039), CD3 eFluor 450 (Cat# 48-0038), CD4 PE-Cyanine7 (Cat# 25-0049), CD4 PerCP-Cyanine5.5 (Cat# 560650, BD Pharmingen), Foxp3 APC (Cat# 17-4777), Foxp3 PE (Cat# 12-4776), CD45RA PE (Cat# 130-092-248, Miltenyi), CD45RA APC (Cat# 130-092-249, Miltenyi), CD62L APC (Cat# 17-0629), CD25 PE (Cat# 130-091-024, Miltenyi), CD25 APC-H7 (Cat# 560225, BD Pharmingen), CD8 PE-Cyanine7 (Cat# 25-0088), Perforin PE (Cat# 12-9994), granzyme B PE (Cat# 12-8899), CCR8 APC (Cat# FAB1429A, R&D System), CD4 FITC (Cat# 11-0049), CD45 APC (Cat# 17-9459), CD14 PE (Cat# 12-0149), CD45RO PE (Cat# 12-0457), CD45RO eFluor 450 (Cat# 48-0457), CD127 APC (Cat# 17-1278). PITPNM3 antibody (Cat# NBP1-31070, Novus) was labeled with APC by Abcam APC Conjugation Kit (ab201807, Abcam) according to the manufacturer's instructions. For the intracellular stain, cells were pretreated with Intracellular Fixation and Permeabilization kit (Cat# 88-8824) according to the manufacturer's instructions. All the reagents were from eBioscience unless indicated otherwise. Cells were subsequently analyzed by multicolor flow cytometry (Gallios, Beckman Coulter, China).