Cellfix

Flow cytometric analyses of the transfected ECs, HEK293 and PAE cells was performed to detect CD39 surface expression. Thus, 24 h, 5 days or 7 days after transfection, cells were washed with PBS (w/o Ca⁺/Mg⁺) and split into two 50 μl aliquots. One aliquot of each sample was stained with 5 μl anti-CD39-fluorescein isothiocyanate (FITC) antibody (Abcam; Clone A1) or 5 μl anti-CD39-phycoerythrin (PE) antibody (BD Bioscience) for 20 min at room temperature in the dark, whereas the second sample was incubated with 5 μl PBS (w/o Ca⁺/Mg⁺) as control. After antibody staining, cells were washed and fixed using 250 μl 1x CellFix (BD Biosciences). Flow cytometry was performed within 6 h using a FACScan cytometer (BD Biosciences, USA) and a total of 10.000 events were acquired in each sample.
CD39 expression of HEK293 cells was also analyzed under a fluorescence microscope. Therefore, the cells were fixed with 500 μl 1x CellFix (BD Biosciences) and incubated for 15 min at room temperature. Afterwards, cells were washed twice and stained with 10 μl anti-CD39-FITC antibody (Abcam, Clone A1) diluted in 500 μl PBS for 20 min at room temperature. Again, the cells were washed twice with PBS and fluorescence microscopic analyses were performed using an inverse microscope (Axiovert 135, Zeiss, Germany).

Whole blood (500 μl/tube) was exposed to a stimulus known to induce piecemeal degranulation—100 ng/ml exotoxin (CCL11)(BioLegend, San Diego, CA), for 2 or 5 h at 37°C. Untreated samples were also included. After incubation, 200 μl of blood was removed, red blood cells were lysed, and the sample was then fixed (BD CellFix™, Becton Dickinson, Belgium).