Cd86 pe clone it2

The expression of a panel of cell surface markers was analyzed using a FACS Calibur (BD Biosciences, San Diego, USA). After harvesting the cells, antibody incubation steps were carried out at 4°C for 30 min in PBS + 1% BSA + 0.1% sodium azide + 1% rabbit serum. Dead cells were excluded using viability marker SYTOX (ThermoFisher). Antibodies used were CD68-FITC (EBM11, DAKO, Denmark), CD86-PE (Clone IT2.2, BD Biosciences), CD163-PERCP-CY5.5 (clone GHI/61, Biolegend, San Diego, USA), CD14 APC-AF750 (clone RMO52, Beckman Coulter, Brea, USA), CD206-PC-7 (clone 3.29B1.10, Beckman Coulter) and CD16-APC (clone 3G8, Life technologies, Frederick, USA).
For FACS-based cell cycle analysis, cells were incubated for 30 minutes with 1μM BrdU at 37°C. BrdU-positive cells were detected with an anti-BrdU-FITC antibody (BD Bioscience) according to the manufacturer's protocol. For determination of DNA content 10μg/ml propidium iodide (PI) was added in the presence of 250 μg/ml RNase.

Single-cell suspensions from the BM and peripheral blood were first incubated with human BD Fc block (BD Biosciences) to block Fcγ receptor and then stained with the following anti-human Abs: CD19-APC (clone HIB19, BioLegend), CD34-PE (clone 563, BD Biosciences), CD20-FITC (clone 2H7, BioLegend), CD27-APC/Cyanine7 (clone O323, BioLegend), IgD-PE (clone IA6-2, BD Biosciences), IgM-FITC (clone G20-127, BD Biosciences), CD43-PE (clone CD43-10G7, BioLegend), CD38-PE/Cy7 (clone HIT2, BioLegend), CD38-Percp/Cy5.5 (clone HIT2, BD Biosciences), CD24-PE (clone ML5, BD Biosciences), CD27-V450 (clone M-T271, BD Biosciences), IgD-BV510 (clone IA6-2, BD Biosciences), IgG-PE/Cy7 (clone G18-145, BD Biosciences), Ki-67–PE (clone Ki-67, BioLegend), CD80-FITC (clone L307.4, BD Biosciences), CD86-PE (clone IT2.2, BD Biosciences), HLADR-PE (clone G46-6, BD Biosciences) and CD69-PE/Cy7 (clone FN50, BD Biosciences). 7-AAD Viability Staining Solution (eBioscience, Thermo Fisher Scientific) was used for live versus dead cell discrimination. Samples were analyzed with a FACSVerse flow cytometer (BD Biosciences) using the FACSuite software. Data analysis was performed with FlowJo 10 software (Treestar).

After 6 h of cultivation, the cells were collected, stained with Fixable Viability Stain 780 (BD Biosciences). Monocytes suspended in PBS were incubated with combined antibodies as follows: CD14 (PerCP-Cy5.5, clone M5E2, BD Biosciences, Cat. no. 550787), CD16 (PE-Cy7, clone 3G8; BD Biosciences, Cat. no. 557744), CD45 (APC-Cy7, clone 30-F11; BD Biosciences, Cat. no. 557659), CD86 (PE, clone IT2.2; BD Biosciences, Cat. no. 555665), CD163 (Alexa Fluor 647, clone GHI/61, BD Biosciences, Cat. no. 562669), HLA-DR (V450, clone L243, BD Biosciences, Cat no. 642276), and CD206 (Alexa Fluor 488, clone 15-2, Biolegend, Cat. no. 321114). Antibodies were used according to the manufacturer’s instructions. From each sample, 10,000–100,000 events were acquired using the LRS II Fortessa cytometer (BD Biosciences, San Diego, CA, USA), and the analyses were performed using BD FACSDiva 8.0.1 and FlowJo software version 10.8.1. All the gate strategies are described in Supplementary Material S3. For tSNE analysis, FlowJo software version 10.8.1 was used. From the gate of viable monocytes, 5,000 events were automatically extracted from each sample using the plug-in DownSample v.3.3.1, generating a representative population of the sample. All tSNE parameters are described in Supplementary Material S3.