Facscanto 2 flow cytometry system

Left-over blood samples for clinical examination from 32 COVID-19 patients and 37 healthy volunteers were collected for analysis. All the samples were processed in the Clinical Lab of Wuhan Union Hospital based on uniform standard procedure. Briefly, peripheral blood mononuclear cells (PBMCs) and plasma were isolated by density gradient centrifugation. Fresh separated PBMCs were stained for flow cytometry, and plasma samples were used for cytokine detection.
Flow cytometry was performed as described previously (9 (link)). Briefly, 1 × 10⁶ PBMCs were stained with indicated antibodies in the dark at room temperature for 20 min. After several washes, the cells were analyzed within 1 h. All samples were detected by BD FACS Canto II Flow Cytometry System and analyzed with the BD FACS Diva Software. Antibodies used for flow cytometry included FITC-CD3 (clone: HIT3a), PE-PD-1 (clone: EH12.1), PE/Cy7-CD56 (clone: B159), APC-CD244 (clone: 2-69), APC/Cy7-CD45 (clone: 2D1), BV421-CD16 (clone: 3G8), BV421-CD4 (clone: RPA-T4), PerCP-CD8 (clone: RPA-T8), PE/Cy7-CD27 (clone: M-T271), and all of these were purchased from BD Pharmingen.

Freshly isolated aortas were resuspended in FACS buffer (PBS containing 1% FBS and 2 mM EDTA) and stained with conjugated antibody for 20 to 30 min at 4 °C. Cells were washed and resuspended in FACS buffer for flow cytometric analyses in which inflammatory cell populations were designated, following gating/stratification of their marker profile. The aortas were cut into small pieces and digested in an enzyme mixture containing 450 U/mL collagenase I (C0130, Sigma‒Aldrich), 125 U/mL collagenase XI (C7657, Sigma‒Aldrich), 60 U DNase I (DN25, Sigma‒Aldrich), and 60 U/mL hyaluronidase (2592, Worthington Laboratories) in PBS with Ca²⁺/Mg²⁺ for 60 min at 37 °C with gentle shaking. After incubation, the digestion mixture was homogenized through a 70-μm nylon mesh. The digestion mixture was centrifuged at 2000 rpm for 15 min at 4 °C, and the cells were simultaneously stained with antibodies at 4 °C for 15 min and then washed and resuspended in a staining buffer. All antibodies are listed in Supplementary Table 3. The cell suspensions were analyzed with a BD FACSCANTO II flow cytometry system (BD Biosciences), and postacquisition analysis was performed with FlowJo7 software (Tree Star).

Anti‐rat CD4 microbeads (Miltenyi Biotec) were employed for the positive selection of CD4⁺ cells from single‐cell suspensions of spleens and kidney grafts in the Isograft, Allograft, and Allograft + sEVs groups. CD4⁺T cells were then incubated with anti‐rat CD25 APC (Biolegend) and anti‐rat Foxp3 FITC antibodies (eBioscience). Multiple‐color flow cytometric analysis was conducted to determine the proportion of CD25⁺Foxp3⁺ Treg cells in CD4⁺T cells using the BD FACSCanto™ II Flow Cytometry System.

Serum was separated from the collected blood samples via centrifugation at 3,000 g for 15 min at 4°C. Thereafter, serum AST and ALT levels were determined using a FUJI DRI-CHEM NX500iVC biochemical analyzer with their respective FUJI DRI-CHEM slide kits (FujiFilm Corporation, Tokyo, Japan). In addition, serum levels of inflammatory cytokines, including interleukin (IL)-6, IL-10, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-12p70, were detected using the BD™ Cytometric Bead Array kit and the BD FACSCanto II flow cytometry system (BD Biosciences, San Jose, CA, United States).

To measure cellular ROS levels, the cells were washed in PBS and incubated with 10 μM of dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime) at 37 °C for 30 min. Cells were treated with Rosup as a positive control. Treated cells were washed in PBS, trypsinized and collected in pre-cooled PBS then analyzed on a BD FACSCanto II flow cytometry system (BD Biosciences). The data were analyzed using FlowJo V10 software, as previously described (37 (link)).