Alexa flour 594 conjugated phalloidin primary antibodies

Platelets activation and adhesion assays were performed as previously described [21] . Brie y, glass-based wells of 24 well plate were coated with cell-free NETs after overnight incubation with corresponding medium at 4℃ in a humidi ed chamber. For controls, 1% of denatured bovine serum albumin (dBSA) was used. Platelets suspension (1×10 7 cells/ml) was then seeded in the wells, cultured for 1 h at 37℃ under 5% CO 2 , xed for 15 min at RT with 4% paraformaldehyde and washed three times using 1×PBS before 20 min permeabilization using 0.1% Triton-X 100. The platelets were then incubated for 30 min with Alexa Flour 594 conjugated phalloidin primary antibodies (1:300, ThermoFisher, Waltham, USA). To assess PS and P-selectin expression, the platelets were stimulated by NETs for 1 h, and the cells were rstly stained with FITC-conjugated-lactadherin (Haematologic, EssexJunction, VT) for 30min, and then stained with primary rabbit anti-P-selectin (1:200, Proteinteck, China), and mouse anti-CD41 (1:500, Novus, USA) antibodies, imaging were observed and photographed using confocal microscopy.

HUVECs were incubated for 4 h in 24 well plates with cell-free NETs or PBS. The cells were xed in 4% paraformaldehyde for 15 min at RT, washed three times using 1×PBS and thereafter blocked for 1h using 10% goat serum with 1% BSA solution in PBS. For detection of TF expression, ECs were incubated overnight at 4℃ with rabbit anti-TF (1:500, ab228968, Cambridge, UK) and mouse anti-CD31 (1:500, ab9498, Cambridge, UK) primary antibodies. Then cells were washed with PBS and re-incubated for 1h at RT with Alexa Fluor 594 conjugated (Proteinteck, China) goat anti-rabbit and Alexa Fluor 488 (Proteinteck, China) conjugated goat anti-mouse secondary antibodies. For detection of intercellular junctions of cells, incubated overnight at 4℃ with rabbit anti-VE-cadherin (1:500, ab33168, Cambridge, UK) primary antibodies followed the Alexa Fluor 488 (Proteinteck, China) conjugated goat anti-rabbit secondary antibodies and further incubated with Alexa Flour 594 conjugated phalloidin primary antibodies (1:300, ThermoFisher, Waltham, USA). They were then stained with 4',6-diamidino-2-phenylindole (DAPI) and xed with mounting medium (Solarbio, Beijing, China) for 5min at RT in the dark. The cells were then observed and photographed using a confocal microscope. The photos were analyzed using Image J software.