Cannabis Modulates Inflammatory Cytokine Levels

IL-6 and IL-8 levels were determined as described previously^{43 (link)} with the following modifications: A549 cells were plated at 5 × 10⁴ cells per well in DMEM complete media (400 µL) in 24-well cell culture plate. They were allowed to attach and grow at 37 °C in air and 5% CO₂ in a humidified incubator overnight with complete DMEM, and then the media was replaced with serum free DMEM. Cell excitation was performed with 300 ng/mL TNFα. Treatments were performed with cannabis crude extract, fraction or combination of compounds together with 100 µL serum free DMEM. IL-6 and IL-8 secretion levels were analyzed after 4 or 6 h of incubation for A549 or KG1 cell lines, respectively. Supernatant samples were collected and tested using IL-6 and IL-8 ELISA kits (DY206 and DY208 respectively, R&D Systems, Minneapolis, MN, USA). Dexamethasone was used as a positive control. For cell viability, an Alamar Blue (resazurin) assay was performed on each well as described previously^{43 (link)}. For dose response assays, data points were connected by non-linear regression lines of the sigmoidal dose–response relation. GraphPad Prism version 6.1 (https://www.graphpad.com/scientific-software/prism/, GraphPad Software Inc., San Diego, USA) was employed to produce dose–response curves and IC50 doses were calculated using nonlinear regression analysis.