Anti cd11c apc hl3

Lipopolysaccharide (LPS) (Escherichia coli, E. coli 055:B5) and PMA [phorbol 12-myristate 13-acetate ≥ 99% (TLC), film or powder] were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ionomycin (Free Base) was purchased from MKbio (Shanghai, China). Recombinant mouse GM-CSF (granulocyte-macrophage colony stimulating factor), IL-4 (interleukin 4) and IL-2 (interleukin 2) were obtained from Peprotech (Rocky Hill, NJ, USA). Cell staining was performed by using the following mAbs from BD Biosciences (Franklin Lakes, NJ, USA): anti-CD45 FITC (30-F11, #553079), anti-CD11b PerCP-Cy5.5 (M1/70, #550993), anti-CD11c APC (HL3, #550261), anti-F4/80 BV421 (T45-2342, #565411), anti-Ly-6G (Gr-1) PE (1A8, #551461), anti-CD80 PE (16-10A1, #553769), anti-MHC II BV421 (M5/114.15.2, #562564), anti-CD86 PE (GL1, #561963), anti-CD40 BV421 (3/23, #562846), anti-CD3 FITC (17A2, #561798), CD4 PerCP-Cy5.5 (RM4-5, #550954), anti-CD8a PerCP-Cy5.5 (53-6.7, #551162), anti-IL-2 PE (JES6-5H4, #554428), anti-TNFα BV421(MP6-XT22, #563387) and anti-IFNγ APC (XMG1.2, 554413). The isotype-matched mAbs for control staining were from BD Biosciences. Cytotoxicity LDH Assay Kit-WST was purchased from Dokindo (Tokyo, Japan).

Blood samples and splenocytes from anti-CD25- or IL2-treated mice and relevant controls were stained with anti-CD3 FITC (145-2C11, BD Biosciences), anti-CD4 APC-Cy7 (GK 1.5, BD Biosciences), anti-CD8a PerCP-Cy5.5 (53–6.7, BD Biosciences), anti-CD25 APC (PC61, BD Biosciences), anti-CD44 BV421 (IM7, BD Biosciences) and anti-CD62L BV510 (MEL-14, BD Biosciences) antibodies. Cells were then fixed and permeabilized using the Foxp3/Transcription factor fixation/permeabilization Kit (Invitrogen). Cells were then additionally stained with anti-Foxp3 Alexa700 (FJK-16 s, eBioscience), anti-T-bet PE (4B10, BD Biosciences), anti-RORγT PE-CF594 (Q31-378, BD Biosciences) and anti-GATA3 PE-Cy7 (L50-823, BD Biosciences) antibodies. Alternative staining consisted in anti-CD3 FITC (145-2C11, BD Biosciences), anti-TCRγδ PE-CF594 (GL3, BD Biosciences), anti-CD19 BV421 (1D3, BD Biosciences), anti-NK1.1 Alexa700 (PK136, BD Biosciences), anti-NKp46 PE (REA815, Miltenyi), anti-CD11b PE-Cy7 (M1/70, BD Biosciences), anti-CD11c APC (HL3, BD Biosciences) and anti-Ly6G APC-Cy7 (1A8, BD Biosciences) antibodies. Cells were then fixed using CellFix reagent (BD Biosciences). All samples were processed with a Gallios flow cytometer (Beckman Coulter) and the data analyzed using the Kaluza software (Beckman Coulter).