Ab100549

The levels of IL‐10 and IL‐12 in the supernatant of THP‐1 cells treated with/without MHCC97‐H cell‐derived exosomes or PBS were determined using ELISA kits (ab100549 and ab46035; Abcam). Briefly, centrifugation at 1000×g for 5 min was performed at 4°C to collect the supernatant, 100 µL of which was added into precoated 96‐well plates. After incubation at room temperature for 2.5 h, cells were subjected to rinse four times with wash solution, and 60 min further incubation at room temperature with addition of 100 µL Biotin‐labeled antibodies. The treated cells underwent wash gain and treatment with 100 µL horseradish peroxidase (HRP)‐conjugated streptavidin at 37°C for 45 min. A 30 min color development was performed on the cells with TMB substrate solution in the dark after rinsing. Stop solution was later added to stop reaction. Lastly, the color intensity was measured at a wavelength of 450 nm by a microplate reader (PHERAstar FSX; BMG LABTECH).

Plasma levels of IL-35, IL-10, IL-6, IL-17, TNFα, and IL-1β cytokines concentrations in 62 COVID-19 patients and 18 healthy controls were determined using commercially available ELISA kits (Human IL-35, NBP3-06774, Novus Biologicals; human IL-10, ab100549, Abcam; human IL-6, DY206-05, R&D; human IL-17, DY317-05, R&D; human TNFα, ab181421, Abcam; human IL-1b, DY201-05, R&D). Assays were performed strictly following the manufacturer’s instructions. All samples were measured in duplicate.

Cells in the logarithmic growth phase were detached using 0.25% trypsin-0.02% EDTA. After centrifugation, the cells were resuspended in complete culture medium and then subjected to incubation in a 24-well plate at a density of 6 × 10⁵ cells/well at 37° C in a 5% CO₂ atmosphere. After the cells adhered to the well, the cell supernatant was collected and stored at -20° C. The levels of ICAM-1 (ab2213, Abcam, Cambridge, UK), IL-6 (ab6672, Abcam, Cambridge, UK), IL-10 (ab100549, Abcam, Cambridge, UK), and IL-1β (ab214025, Abcam, Cambridge, UK) in the cell supernatant were detected in strict accordance with the provided instructions of the ELISA kit. The experiment was repeated three times to calculate the mean values.