Legendplex human inflammation panel

Cytokines were quantified using the LEGENDPLEX Human Inflammation Panel (BioLegend, San Diego, CA, USA), following the manufacturer’s instructions. Panels containing a fluorescent dye, allophycocyanin (APC)-labeled beads conjugated to a monoclonal antibody specific for each target cytokine, were used to quantify IL-1β, IFN-γ, TNF-α, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-23, and IL-33, as well as MCP-1 (CCL2) and IL-8 (CXCL8) chemokines. The panel detects cytokines with a high sensitivity of 0.6–2.1 pg/mL. Data acquisition was performed with a BD FACS Calibur dual-laser flow cytometer (BD Biosciences, San Diego, CA, USA) at the Laboratorio Nacional de Citometría de Flujo, Instituto de Investigaciones Biomédicas, UNAM. Flow cytometry data were analyzed with the LEGENDPLEX software v7.0 (BioLegend) to obtain the concentration of each cytokine in the samples. Assays were performed in duplicates.

The immunological analysis consisted of the determination of proinflammatory cytokine levels (IL-1β, IL-6, IL-12, IL-17A, IL-18, IL-23, IL-33, TNF-α, INF-α, and INF-γ), chemokines IL-8 and monocyte chemoattractant protein-1 (MCP-1), and immunoregulatory cytokines IL-10 and TGF-β in GCF and serum. After thawing, both sera and GCF were centrifuged at 5800× g to remove small clots, plaque, and cellular elements. The levels of all cytokines, except for TGF-β, were determined by multiplex bead analysis using a flow cytometer (Attune, ThermoFisher Scientific, New Castle, DE, USA) and commercial immunoassay kit (LEGENDplex^™ Human Inflammation Panel, BioLegend, San Diego, CA, USA) according to the manufacturer’s recommended protocol. TGF-β was analyzed by the enzyme-linked immunosorbent assay (ELISA) (DuoSet ELISA kit, R&D Systems Inc, Minneapolis, MN, USA). All samples were analyzed in duplicates, and mean values were used. Variations between duplicates were less than 12%. The analyses from each of the two study periods were performed simultaneously and under identical experimental conditions. The results were expressed as the mean concentration of cytokines in serum (pg/mL) or pg/30 s (GCF).

Extracted exosomes 10 μg were incubated with 1 µl Aldehyde/Sulfate Latex Beads (Invitrogen) 15 min at RT. After washed, exosomes-beads complex were incubated overnight with anti-human TSP-1 antibody (Proteintech). Complex were washed with PBS and incubated with the secondary antibody (Biolegend) for 1 h at 4°C. The expression of TSP-1 of complex were detected by BD celesta flow cytometer.
After treatment with 10 μg/ml exosomes for 12 h, culture supernatant was collected. Inflammation cytokines secreted by THP-1 derived macrophages and PBMC derived macrophages and CD4⁺T cell were detected with LEGENDplex™ Human Inflammation Panel (Biolegend), following the manufacturer’s instructions. The Panel allowed for simultaneous quantification of 13 human inflammation cytokines: IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IL-18, IL-23 and IL-33. Inflammation cytokines were measured on the BD celesta flow cytometer.

RPMI and FBS were obtained from Thermo Fisher Scientific (Waltham, MA, USA), DNA-Prep Reagent Kit from Beckman Coulter (Brea, CA, USA), NADPH from Roche (Mannheim, Germany), Legendplex™ Human Inflammation Panel from BioLegend (San Diego, CA, USA) and MILLIPLEX^® MAP Kit and Superoxide Dismutase Assay kit II from Merck (Kenilworth, NJ, USA). All other chemicals were purchased from Sigma-Aldrich (Saint-Louis, MO, USA).

Cells were seeded in 24-well plates at day six at a density of 4x10⁵ cells/well in a volume of 500 μL medium for 24 hours before the start of experiments. Cells were exposed to 100 μM, 500 μM or 1000 μM of CoCl₂ to investigate the inflammatory response in HaCaT cells following exposure to CoCl₂. Supernatants were collected after 30 minutes, 24 hours and 48 hours. Exposure time 30 minutes function as a model control time. Supernatants were centrifuged and aliquoted and stored at—80°C until analysis. Experiments were performed five times with three technical replicates.
Cytokine/chemokine production was measured using the immunoassay LEGENDplex Human Inflammation Panel from BioLegend (San Diego, CA) analyzing IFN-α, IFN-γ, TNF, CCL2, IL-6, CXCL8, IL-10, IL-12p70 IL-17A, and IL-23 according to manufacturer instructions using an Accuri C6 (Becton Dickinson, San Jose, CA).

Cell expansion was determined using a Cell Counting Kit-8 [24 (link)] (CCK8; Beyotime, Shanghai, China). NK cells were treated with calcitriol for 48 h and the supernatant was collected for the Cytokine-releasing assay. The cytokine levels were examined using the LEGEND Plex Human Inflammation Panel (BioLegend, California, USA). First, the cytokine capture beads were incubated with the standards or samples and then further incubated with biotinylated detection antibodies. The biotinylated detection antibody-binding solution, streptavidin (SA)-PE, was subsequently added to provide the fluorescent signal [25 (link)]. These signals were then analyzed using the FACS assay.
Cell cycle was measured using a Cell Cycle Detection Kit (Beyotime, Shanghai, China). NK cells were treated with calcitriol for 72 h. These cells were fixed in 70% ice-cold ethanol and then dyed with ribonuclease A (RNase A) and propidium iodide (PI) (Beyotime, Shanghai, China) [26 (link)].