Human inflammatory cytokine cytometric bead array cba

MDA-MB-231-derived CSCs were seeded in an ultra-low attachment 6-well plate. After 6 days, the cells were treated with 1 µM (+)-JQ1, 0.1 µM ARV825, 2 µM Verteporfin, 50 µM calcitriol, and 20 µM caffeic acid for 24 h. The cytokine profiling of cells was assessed in a supernatant culture medium using Human Inflammatory Cytokine Cytometric Bead Array (CBA) (BD Biosciences) and FACS. The procedures followed the manufacturer’s protocol. The samples were measured by flow cytometry (Accuri C6, BD Biosciences). CBA data were analyzed and quantitated using BD FCAP array software.

LPS was purchased from Sigma Aldrich (St Louis, MO, USA) and originated from S. Typhimurium (LPS1) and E. Coli (LPS2), respectively. All stimulations were performed for a total of 6 h except for rhS100A9 (20 h). IL-1β and HMGB1 was from R&D Systems. Recombinant human S100A9 (rhS100A9) was a gift from Active Biotech AB and a detailed description on endotoxin-free S100A9 generation and purification has been published previously [15 (link)] and was used in the presence of calcium and zinc (Ca²⁺ ≥200 μM; 10 μM ZnCl₂ [34 , 35 (link)]). Supernatants from stimulated or siRNA transfected cells were harvested and analyzed using human inflammatory cytokine cytometric bead array (CBA; BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions or using IL-6 and IL-8 Quantikine ELISA (R&D Systems, Minneapolis, MN, USA). Annexin V-allophycocyanin (APC) and propium iodide (PI) staining was performed according to the manufacturer’s instructions (BD Biosciences). The cycloheximide (CHX) experiments (Sigma Aldrich) where performed by adding 10 μg/ml CHX, with or without 100 ng/ml LPS for 6 h.

Human study 1 was performed in the Integrated Critical Care Unit at South Tyneside and Sunderland Foundation Trust, supervised by a critical care physician.
Ethical approval was granted by the Yorkshire and the Humber–South Yorkshire Research Ethics Committee (17/YH/0021), and the study was sponsored by Newcastle upon Tyne Hospitals National Health System (NHS) Foundation Trust. Ten healthy male volunteers (mean age 25 y, range 18 to 25) gave informed, written consent to receive intravenous administration of LPS (Cat# 94332B1, donated by the NIH) and injected intravenously as a bolus dose of 2 ng/kg. Baseline measurements were undertaken between 8:00 and 10:30. Human cortisol was measured using a chemiluminescent immunoassay (Cat# 313261, DiaSorin S.p.A.). Human GDF15 was measured using an electrochemiluminescent immunoassay on the MesoScale Discovery platform with antibodies and standards from R&D Systems Europe. Cytokine concentrations were measured using a human inflammatory cytokine cytometric bead array (CBA) (Cat# 551811, BD Biosciences).