CD31-FITC, 5.6E, 2 mL, ASR

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Endothelial cells were stimulated with P. aeruginosa PAO1 strain and QSSMs (20 µM) for 20 hours and the expression of cell adhesion and inflammatory markers was assessed by flow cytometry (Gallios, Beckman-Coulter) using 1 × 105 endothelial cells stained	Endothelial cells were detached using trypsin (Sigma-Aldrich, USA) and washed in PBS. Cells were then incubated with the primary antibodies at room temperature in the dark for 30 min. Further, the cells were washed twice and centrifuged at 400g, 10 min, in PBS suplemented with 1% BSA.	Flow cytometry data were analyzed using the Gallios software (Beckman-Coulter).

Upstream tips
Endothelial cells were stimulated with P. aeruginosa PAO1 strain and QSSMs (20 µM) for 20 hours and the expression of cell adhesion and inflammatory markers was assessed by flow cytometry (Gallios, Beckman-Coulter) using 1 × 105 endothelial cells stained
Protocol tips
Endothelial cells were detached using trypsin (Sigma-Aldrich, USA) and washed in PBS. Cells were then incubated with the primary antibodies at room temperature in the dark for 30 min. Further, the cells were washed twice and centrifuged at 400g, 10 min, in PBS suplemented with 1% BSA.
Downstream tips
Flow cytometry data were analyzed using the Gallios software (Beckman-Coulter).

Publication protocol

Endothelial cells were stimulated with P. aeruginosa PAO1 strain and QSSMs (20 µM) for 20 hours and the expression of cell adhesion and inflammatory markers was assessed by flow cytometry (Gallios, Beckman-Coulter) using 1 × 105 endothelial cells stained with fluorochrome-conjugated (FITC - fluorescein-isothiocyanate and PE - phycoerythrin) primary antibodies against IL-1β, IL-6, ICAM-1 PECAM-1, P-selectin, VE-cadherin (Beckman-Coulter). Endothelial cells were detached using trypsin (Sigma-Aldrich, USA) and washed in PBS. Cells were then incubated with the primary antibodies at room temperature in the dark for 30 min. Further, the cells were washed twice and centrifuged at 400g, 10 min, in PBS suplemented with 1% BSA. For negative controls, endothelial cells were stained with the corresponding isotype-matched IgG antibodies (Beckman-Coulter). Flow cytometry data were analyzed using the Gallios software (Beckman-Coulter).

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