Cd14 af700

For immunofluorescence staining of whole blood and PBMC, antibodies included CD45-ECD (Beckman Coulter, Indianapolis IN), CD3-e780, CD4-e450, CD8-AF700, CD15-FITC (eBioscience, San Diego, CA), CD45-FITC, CD4-PerCP-Cy5-5, CD14-PECy7, HLA-DR-APC, HLA-DR-BV421, CD8-APC-H7, CCR7-BV605, CD25-BV605, CD45RA-PECy7, CD45RO-APC-H7, CCR4-PECy7, CD38-APC, CD127-BV650 (BD Bioscience, San Jose, CA), CD14-AF700, and CD3-AF700 (Biolegend, San Diego, CA). For phosphoSTAT staining, antibodies included CD3-BV785 (Biolegend), CD4-BV605, CD8-BV510, CD14-PECy7, CD19-BV421, CD16-BV650, pSTAT3-647, pSTAT5-PE (BD Biosciences), and pSTAT1-488 (Cell Signaling Technologies, Danvers, MA). Cell types were identified according to the criteria of the Human Immunology Project [19 (link)], and a full list of antibodies used to identify individual cell types including those not reported here can be found in Additional file 2: Table S1. Hematological toxicity was graded according to NCI CTCAE v4.0 guidelines and is reported in Additional files 3 and 4: Tables S2 and S3.

Viable frozen peripheral blood samples from 2 DS patients with DS-associated myeloid preleukemia were obtained from the biobank commission of the Princess Máxima Center for Pediatric Oncology. Mononuclear cells were stained with a cocktail of the following antibodies: CD3-BV650 (Biolegend, Clone UCHT1, 300467, 1:100), CD4-PerCP/Cy5.5 (Biolegend, Clone OKT4, 317427, 1:200), CD8-BV785 (Biolegend, Clone SK1, 344739, 1:100), CD19-BV421 (Biolegend Clone HCD14, 30224, 1:100) , CD14-AF700 (Biolegend, Clone HCD14, 325614, 1:100), CD56-BV711 (Biolegend, Clone HCD56, 318335, 1:50), CD34-APC (Biolegend, Clone 561, 343607, 1:50), CD38-PE (Biolegend, Clone HIT2, 303505, 1:50) , CD33-PE/Cy7 (Biolegend, Clone WM53, 303433, 1:100), CD117-PE-dazzle594 (Biolegend, Clone 104D2, 1:100), CD16-FITC (Biolegend, Clone 3G8, 302005, 1:100), CD20-FITC (Biolegend, Clone IVB201, 302303, 1:100). Bulk T-cells (CD3+/CD4+ and CD3+/CD8+) and preleukemic blast cells were sorted with the Astrios-EQ. Preleukemic blast cells were sorted according to the diagnostics flow data. Cell pellets were used for DNA isolation. Public data was used for the other 4 myeloid preleukemia samples^{11 (link)}.

Lidocain (4 ng/mL) was added to macrophages and then cells were detached by gentle scraping, spun down, and collected in buffer (PBS plus 0.5% bovine serum albumin). Cells were incubated for 20 min at 4°C with appropriate antibodies, fluorescence minus one (FMO), and single stains were taken along for every antibody. Antibodies were as follows: CD14-AF700, CD68-PE-CY7 CD86-BV650, CD163-BV421, CD209-FITC, CD206-APC (Biolegend, San Diego, CA, USA). The cells were then washed twice with 100 μl of flow buffer, centrifuged, and resuspended in 200 μl flow buffer for analysis. Flow cytometry was performed with fluorescence-activated cell sorter (FACS) Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The cell populations were gated based upon FSC and SSC parameters and normalized to cells alone (without antibody) to adjust for cell-specific autofluorescence and a gate was set on the CD14 positive population (Supplementary Figure 4). Data were analyzed using FlowJo (FlowJo JCC, Ashland, OR, USA).