Canto 2 cytofluorometer

The membrane expression of E-cad on PBMCs was investigated using flow cytometry. Cells were analyzed according to fluorescence intensity for CD3, CD14, CD16, and CD20, markers. MAb anti-CD3-FITC, anti-CD20-PC5, anti-CD16-PE, and anti-CD14-FITC were purchased from Beckman (Beckman coulter, Villepinte, France). Cell surface expression of E-cad was assayed using an anti-E-cad mAb-APC. Fluorescence intensity was measured using a Canto II cytofluorometer (Becton Dickinson, Biosciences, Le Pont de Claix, France), and the results were analyzed using a FlowJo software X.10.0.7.

BeWo cells (1 × 10⁶ cells/well) were cultured in flat-bottom six-well plates and were then incubated in a 5% CO₂ atmosphere for 24 h with 1.6 μg/mL of the CoxbHtrA recombinant protein. Cells were exposed to 2 mM EDTA in PBS for 15 min at 4 °C to release the adherent cells from the culture plate. After centrifugation at 500× g for 5 min, the pellet was suspended in a FACS buffer (2 mM EDTA, 10% FBS in PBS) and was then incubated (1:1000 dilution) for 1 h with a mouse anti-human E-cad ectodomain antibody (HECD-131700, 1:1000 dilution; Life Technologies). After two washes, cells were incubated with a goat anti-mouse IgG and Alexa Fluor-488 secondary antibody. Fluorescence intensity was measured using a Canto II cytofluorometer (Becton Dickinson/Biosciences, le Pont de Claix, France), and the results were analyzed using a FlowJo V10.7.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).

Flow cytometry was used to study the expression of ACE2 on the surface of cells. To do so, Vero E6 cells were cultured in flat-bottom six-well plates at an initial concentration of 1×10⁶ cells/well and were then treated with the drugs. Cells were then detached from the solid support using 2 mM of EDTA in PBS with an incubation period of 15 minutes at 4°C, followed by centrifugation of 500 g for five minutes. The pellet was resuspended in a saturation buffer (2mM EDTA, 10% FSB in PBS) for 30 minutes. For ACE2 labelling, an anti-ACE2-PE mAb (R&D Systems, Minneapolis, USA) was incubated with cells at 4°C in the dark for 30 minutes. Fluorescence intensity was measured using a Canto II cytofluorometer (Becton Dickinson, Biosciences, Le Pont de Claix, France). The results were further analysed using a BD FACSDiva Software v.6.1.3 (Becton, Dickinson and Company, New Jersey, USA).