Legendplex human inflammation panel 1

MCF7 cells were cultured in different media for indicated number of days, and supernatant culture media were collected every 4 days, centrifuged, and stored at −80 °C until analysis. IL‐6 levels of culture supernatants were determined using the LEGENDplex™ Human Inflammation Panel I (BioLegend, 740809), according to the manufacturer's instructions. Briefly, supernatants (50 μL per sample) were incubated with capture beads for 2 h at room temperature on an orbital shaker. Next, detection antibody was added and beads were incubated for 1 h at room temperature. After washing the beads, samples were measured using a BD LSRFortessa™ flow cytometer (BD Biosciences). Cytokine concentration was calculated based on a standard curve using BioLegend's LEGENDplex™ data analysis software.

The LEGENDplex Human Inflammation Panel I (Biolegend, London, UK ) was used to simultaneously quantify 13 human cytokines/chemokines (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) in biopsy tissue lysates according to the manufacturer’s instructions and analysed on a BD LSR Fortessa using the PE and APC channels. Data were analysed using the LEGENDplex data analysis software (Biolegend, London, UK). All cytokines/chemokines were detectable apart from IFN-α2 which was below detection limits.

Human cytokine levels were analyzed using the LEGENDplex^TM human Inflammation Panel 1 (Biolegend, San Diego, CA, USA). A 96-well V-bottom plate was loaded with duplicates, with 25 µL for each sample or standard. 25 µL of mixed beads and 25 µL of assay buffer were added to each sample well. To each standard well, 25 µL of mixed beads and 25 µL of Matrix B were added. The plate was sealed, covered in foil and shaken at 800 rpm for 2 h at room temperature. Then, the plate was centrifuged at 1050 rpm for 5 min, the supernatant was discarded and 25 µL of detection antibody were added to each well. The plate was sealed, covered in foil and shaken at 800 rpm for 1 h at room temperature. 25 µL of Streptavidin-Phycoerythrin (PE) were added directly to each well. The plate was sealed, covered in foil and shaken at 800 rpm for 30 min at room temperature. After centrifugation at 1050 rpm, the supernatant was discarded and 200 µL of wash buffer were added to each well and incubated for 1 min. The plate was centrifuged at 1050 rpm, and the supernatant was discarded and resuspended in 100 µL of 1X wash buffer. The samples were acquired on a Cytoflex S (Beckman Coulter, Brea, CA, USA) and were analyzed with the Legendplex 8.0 Software (Biolegend, San Diego, CA, USA).