Cd41 apc cy7

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).