Cd61 apc

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.

For MK and platelet experiments, 15–25 ml of whole blood was collected by venipuncture from two healthy controls, P3, P4 and P5 in the presence of 1.6 mg/ml EDTA. As access to patient blood was limited, these experiments were performed once (n = 1), with all samples processed in parallel. MKs were differentiated from whole blood samples using a protocol adapted from Balduini et al.^{66 (link)} Briefly, PBMCs were isolated from using Lymphosep (C C Pro, Oberdorla, Germany). Up to 2 × 10⁶ PBMCs were seeded per well in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) with StemSpan ACF medium (StemCell, Vancouver, Canada) supplemented with 10 ng/ml TPO, FLT3-L, IL-6, and IL-11 (all cytokines from Peprotech, Hamburg, Germany). On day 4, APEL2 medium (StemCell) supplemented with 5% Protein-free Hybridoma Medium II (PFHMII, Gibco by Life Technologies, Darmstadt, Germany) and the same cytokine composition was used. On days 1 or 2, 7, 10, and 14, cell morphology was assessed by microscopy and phenotype was analyzed by flow cytometry. For flow cytometry, cells were blocked with FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with CD42a-PE (BD Biosciences, Heidelberg, Germany), CD41-APC/Cy7, and CD61-APC (both Biolegend, San Diego, USA). Analysis was performed using a FACS Canto II and FACSDiva software v8.0.1 (BD Biosciences).