I ab fitc

The following directly labeled anti-mouse monoclonal antibodies were used for BM cell phenotypical analysis: CD90.2-APC and CD45-PE/Cy7 for lymphoid progenitors, CD61-APC and CD41-FITC for megakaryocytic population, CD71-PE and Ter119-FITC for erythroid precursors, CD11b-PE and Gr1-FITC for granulocytes/monocytes progenitors, Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-FITC, Sca1-PE, cKit (CD117)-APC for hematopoietic stem cells, all purchased from BioLegend (BioLegend, San Diego, CA, USA).
The phenotypical analysis of splenocytes was performed using the following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE, I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was used.
Single-cell suspensions of splenocytes or BM cells were incubated with the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C for 20 min for cell surface staining. For intracellular staining (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer (eBioscience), according to the manufacturer’s instructions. Measurements were performed with a FACSCalibur flow cytometer as described above.

The phenotypical analysis of splenic DCs was performed using the following antimouse antibodies: CD11c-PE, CD11c-APC, and I-Ab-FITC (Biolegend, San Diego, CA, USA) for the identification of the splenic DC population; CD40-PECy5, CD80-PECy5- CD86-PECy5, B7-H1-APC, and DEC205-AlexaFluor^®647 (Biolegend) for detecting DC-specific cell surface markers; and IL-1α-PE and IL-1β-PE (eBioscience, San Diego, CA, USA) for evaluating intracellular cytokine production by DCs. Single-cell suspensions of splenocytes were incubated with the fluorescently labeled antibodies against cell-surface markers in a staining buffer containing 1% BSA at 4 °C for 20 min. For intracellular cytokine measurements, cells were fixed and permeabilized with the FOXP3 Fix/Perm Buffer Set (BioLegend) and incubated with the corresponding anticytokine antibodies at room temperature for 30 min. All measurements were done with a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed by the CytExpert software (Beckman Coulter).

The following antibodies from eBioscience (San Diego, CA, USA) were used: anti-mouse CD4 APC-H7 (GK1.5), CD25 PE (PC61.5), IL-17 PE (eBio17B7), IL-17 APC (eBio17B7), Foxp3 PE-Cy7 (FJK-16s), IgG 2a PE-Cy7 (eBR2a), CD39 PE (24DMS1), CD73 PE-Cy7 (Ty/11.8), CTLA-4 PE (UC10-4F10-11), Lag-3 APC (C9b7w), CCR9 PE (eBioCW-1.2), α4β7 PE (DATK32), B220 (RA3-6b2), CD19 APCH-7 (1D3), CD11c (N418), CD62L FITC (MEL-14), CD45.1 PE-cy7 (A20) purified anti-CD3 (145-2C11), and purified CD16/32 (93). From Biolegend (San Diego, CA, USA), we used CD4 PECy7 (GK 1.5), I-Ab FITC (25-9-17) and CD11b APC (M1/70), and purified α-IFNγ (XMG1.2). Recombinant mouse IL-2, TGF-β1, IL-6, and IL-1β were purchased from eBioscience. All-trans-retinoic acid, OVA protein, PMA, and ionomycin were purchased from Sigma Aldrich (St. Louis, MO, USA).