Legendplex human inflammation panel 1 13 plex

To analyse cytokine and chemokine levels in plasma and culture supernatants, the following kits were used: LEGENDplex Human Inflammation Panel 1 (13-plex) (Biolegend), Cytometric Bead Array Human Inflammatory Cytokine Kit (BD Biosciences) and Cytometric Bead Array Human Chemokine Kit (BD Biosciences).

To measure the cytokines levels, 25 μL of ascites of OCs was mixed with 25 μL of assay buffer. Then, 25 μL of 13-plex-beads were pipetted to a 96-well microplate (LEGENDplex™ Human Inflammation Panel 1 (13-plex) #740,809, BioLegend, USA). This assay can quantify a total of 13 cytokines/chemokines (the minimum detectable concentration (MDC) in brackets: IL-1β (1.5 + 0.6 pg/ml), IFN-α2 (2.1 + 0.2 pg/ml), IFN-γ (1.3 + 1.0 pg/ml), TNF-α(0.9 + 0.8 pg/ml), MCP-1 (1.1 + 1.2 pg/ml), IL6 (1.5 + 0.7 pg/ml), IL-8 (2.0 + 0.5 pg/ml), IL-10 (2.0 + 0.5 pg/ml), IL-12p70 (2.0 + 0.2 pg/ml), IL17A (0.5 + 0.pg/ml), IL-18 (2.0 + 0.5 pg/ml), IL-23 (1.8 + 0.1 pg/ml), IL-33 (4.4 + 1.5 pg/ml)). The microplate was then incubated and shaken for 2 h at room temperature in which the analytes (cytokines) bound to an antibody-conjugated capture bead. After washing, biotinylated detection antibodies (25 μL) were added and bound to the analytes. Streptavidin–phycoerythrin (25 μL) was subsequently added that bound to the antibodies and provided a fluorescent signal with intensities in proportion to the amount of the bound analyte. After 1 h incubation, beads were washed and flow cytometer was used to quantified the fluorescent signal, and concentrations of the analytes were determined based on a known standard curve using LEGENDplex™ data analysis software (BioLegend, USA).

Supernatants were assessed for cytokine production using the LegendPlex Human Inflammation Panel 1 (13-plex) in a V-bottom plate per the manufacturer’s instructions (BioLegend). Flow cytometry data were acquired on a BD Fortessa analyzer (BD Biosciences) and assessed using FlowJo software (version 10.7, Tree Star). Where indicated, ELISA to detect human IL-8 was done using culture supernatants at 1:100 dilution in a Quantikine ELISA kit (R&D Systems) per manufacturer’s instructions. One sample run by LegendPlex did not have sufficient volume to be further assessed by ELISA.

After sampling, blood was centrifuged to obtain sera as the supernatant. Afterward, sera were diluted 1:2 with a balanced electrolyte crystalloid solution and stored at −20 °C. On the day of analysis, diluted sera were thawed, and cytokine concentrations for IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 were determined using a LEGENDplex™ Human Inflammation Panel 1 (13-plex) as recommended by the manufacturer’s protocol instructions (Biolegend, San Diego, CA, USA) [10 (link),11 (link),12 (link),13 (link)]. In brief, samples were mixed with cytokine-specific capture beads and subsequently incubated first with detection antibodies and then with PE-conjugated detection antibodies (all at room temperature in the dark under shaking), and subjected to flow cytometric analysis. Results were analyzed using Qognit Legendplex Analysis Software (Version 7.1, Biolegend, San Diego, CA, USA).

PEEP, inspiratory pressure (P_insp), and Horovitz index (partial pressure of oxygen [pO₂]/fraction of inspired oxygen [FiO₂]) were defined as key ventilatory parameters. Additionally, the need of vasopressors (noradrenaline) was documented. VWf, ADAMTS13, lactate dehydrogenase and platelets were assessed as markers of thrombotic microangiopathy. C-reactive protein and a cytokine panel were used as markers of inflammation: The LEGENDplex Human Inflammation Panel 1 (13-plex) (BioLegend, CA, USA) containing interleukin (IL)-1β, interferon (IFN)-α2, IFN-γ, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1 (CCL2), IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 was used according to the manufacturer’s instruction. The concentration of the analytes was calculated using the LEGENDplex Cloud-based Data Analysis Software v.2021.07.01.