Xcellence pro image software

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).

Polyploidy of differentiated MKs was analyzed by immunocytochemistry. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD61 (GPIIIa; BD Biosciences) antibody in concentrations recommended by the manufacturer in order to detect the MKs in population, followed by staining with 4′,6-diamidino-2-phenylindole dihydrochloride nucleic acid stain (DAPI; Invitrogen, Carlsbad, CA, USA). Cell images were acquired by fluorescence microscopy using an Olympus IX81 microscope (Olympus, Shinjuku, Japan) and the Xcellence Pro image software (Olympus).
Frequencies of polyploid MK cells in differentiated cell populations were quantified with flow cytometry. MKs were stained with APC-Cy7-conjugated anti-CD41 antibody (GPIIb; BioLegend), washed with phosphate-buffered saline (PBS) and treated with 200 µL of Cytofix/Cytoperm^TM fixation and permeabilization solution (BD Biosciences) for 20 min at room temperature, according to the manufacturer’s protocol. Afterwards, the cells were stained with PI staining solution (10 μg/mL) supplemented with 10 U/mL of RNAse A (Sigma-Aldrich, St. Louis, MO, USA) and acquired with a FACSCanto™ flow cytometry system.

Killing capacity of CAR-Ts was assessed by co-cultivation with CTV-labelled target cells (E:T ratio 2:1) for 48h. Afterwards, 7-AAD (BioLegend) was used to discriminate dead CTV⁺ cells via flow cytometry. For the evaluation of cytotoxicity in co-cultures of CD176-CAR-Ts with Panc-1 cells for 4 days, transmitted-light microscope images were taken with an Olympus IX81 microscope combined with a digital B/W camera using 10x objective lenses and analyzed with Xcellence Pro image software (all from Olympus). Representative pictures are shown.