Digital b w camera

The feasibility to adhere to blood coagulation related stimuli and to accordingly change morphology was assessed with a method designed according to a protocol used by Cho et al.^{46 (link)}. 8 well PCA chamber slides (Sarstedt, Nümbrecht, Germany) were coated over night at 4 °C with 100 µg/ml fibrinogen (Sigma-Aldrich). Platelets were transferred to the coated wells and incubated in the presence of 1 mM ADP and 1 U/ml thrombin (both Sigma-Aldrich) for 90 min at 37 °C. After washing three times with PBS, the PLTs were fixated with Cytofix (BD Biosciences) for 10 min at 37 °C and washed twice with 100 mM glycine (Carl Roth, Karlsruhe, Germany) and once with PBS. The PLTs were permeabilized for 3 min with 0.2% Triton-X-100 (Sigma-Aldrich) at RT, washed three times with PBS and subsequently stained for 20 min at 37 °C with phalloidin-labelled in TexasRed (Invitrogen) diluted 1/70. The cells were washed three times with PBS and once with water. The grid of the PCA slide was detached, mounting solution with DAPI (Dianova, Hamburg, Germany) was applied and the slide was covered with a cover slip. Adhesion of PLTs to fibrinogen was analyzed by fluorescence microscopy using an Olympus IX81 microscope (Olympus) combined with a digital B/W camera (Olympus) and analyzed with Xcellence Pro image software (Olympus).

Capability of MKs to form proPLT was studied as a functional parameter of iPSC-derived MKs, as previously described [14 (link),19 (link)]. Cells harvested at day 19 of differentiation, or cells directly after cryopreservation, were seeded on 12-well suspension culture plates (Greiner CELLSTAR^®; Greiner Bio-One, Kremsmünster, Austria) in APEL medium supplemented with 50 ng/mL of TPO and 50 ng/mL of SCF. After 24 h the images of MKs forming proPLTs were acquired using an Olympus IX81 microscope with a digital B/W camera (Olympus) and analyzed with the Xcellence Pro image software. ProPLTs were detected as elongated uniform tubular structures with periodic PLT-sized swellings [73 (link)].