Fc500 series

As previously our group has described^{17 (link)}, EMV from the culture medium of p-cresol -treated and untreated HUVECs were isolated by ultracentrifugation. The media was centrifuged (Heraeus Labofuge 400R) at 409×g for 5 min at 4 °C to remove any intact cells, followed by centrifugation at 789×g for 10 min at 4 °C to remove cell debris. The media was then transferred to ultracentrifuge 25 × 89 mm polypropylene tubes (Beckman Coulter, Brea, CA, USA) and centrifuged at 18,000×g for 90 min at 10 °C in an Optima XPN-100 ultracentrifuge with a 70Ti rotor (Beckman Coulter). The supernatant containing EMV-free media was removed and the pellets containing EMV were resuspended in PBS sterile and quantified by flow cytometry (FC500 Series, Beckman Coulter). Absolute values of EMV were calculated using the following formula: (EMV counted × standard beads/L)/ standard beads counted (Flow-Count Fluorospheres, Beckman Coulter). Results were expressed as the number of EMV per microliter of culture medium. EMV derived from untreated HUVECs were defined as control EMV (CnEMV) and p-cresol-treated HUVECs were defined as p-cresol EMV (PcEMV).

Cellular ROS levels were measured by flow cytometry, using 2′,7′ dichlorofluorescein diacetate (DCFDA) [19 (link)]. Cells were plated in 6-well plates, and then subjected to the indicated treatments. Negative and positive controls include pretreatment of cells with N-acetyl-l-cysteine (NAC; 1 h) and Tert-Butyl Hydrogen Peroxide (TBHP; 4 h), respectively. Cells were harvested by centrifugation (1000g, 5 min). The pellets were washed twice with PBS, and each sample was incubated with 20 μM DCFDA working solution (30 min, 37 °C in the dark). ROS was detected using FC 500 Series flow cytometer (Beckman Coulter, CA, USA) with excitation and emission spectra of 488 nm and 525 nm, respectively, and analyzed with FlowJo Version X software (Tree Star, San Carlos, CA).

A FACSLyric™ (Becton Dickinson, USA) flow cytometer was employed for cytokine detection, and Beckman Coulter’s FC 500 series (Beckman Coulter, USA) was employed for CBC determination. The Alinity and ARCHITECT Systems were used for IgG levels against SARS-CoV-2, and the EUROIMMUN Analyzer I was used for the ELISA reader. The incubator, centrifuge, vortex mixer and automatic cell counter were from Thermo Fisher Scientific, Inc. (USA). Phosphate-buffered saline (PBS) with a pH of 7.4 was purchased from Sigma‒Aldrich (USA), heparin and EDTA tubes were purchased from Becton Dickinson (USA), and RPMI-1640 medium was purchased from Life Technologies (USA).