Cxcl12 sdf 1α

Cannabinoids used in cytotoxicity and migration experiments were ACEA (CB1 agonist), AM251 (CB1 antagonist, μ-opioid receptor antagonist, GPR55 agonist, ion channel activation), AM630 (CB2 antagonist/inverse agonist, weak partial CB1 agonist, ion channel activation), (-)-cannabidiol (CNB) (GPR55 & weak CB1 antagonist, CB2 inverse agonist, weak agonist at VR1 vanilloid receptors, and modulator at opioid receptors), JWH133 (CB2 antagonist), and R-(+)-methanandamide (RM) (CB1 agonist, activity against GPR and ion channels) purchased from TOCRIS Bioscience (Bristol, UK). Fludarabine (2-Fluoroadenine-9-β-D-arabinofuranoside, SIGMA-ALDRICH, Vienna, Austria) served as control in cytotoxicity experiments, CXCL12/SDF-1α (R&D SYSTEMS, Abingdon, UK) and CXCR4 antagonist AMD3100 octahydrochloride (TOCRIS Bioscience, Bristol, UK) were controls in migration experiments.

To measure the biomarkers, ten millilitres of fasting venous blood were withdrawn from each participant between 8.30 a.m. and 9.30 a.m. at the research centre. The blood was sent to the laboratory within three hours after the first venepuncture. Subsequently, the whole blood samples were centrifuged at 1650 g for 25 min at room temperature to obtain the plasma. All the blood and plasma samples from both groups at the three time-points were stored at −80 °C. The nine plasma biomarkers were examined using commercially available enzyme-linked immunosorbent assay (ELISA) kits, namely, IL-6, IL-1β (ThermoFisher, Waltham, MA, USA), CXCL12/SDF-1α, CXCL5/RANTES (R&D Systems Inc., Minneapolis, MN, USA), BDNF (Promega Corporation, Madison, WI, USA), DHEA (CUSABIO, Houston, TX, USA), cortisol, hs-CRP (Tecan, Männedorf, Switzerland), and sgp130 (RayBiotech Inc., Norcross, GA, USA). Assays for each biomarker were run on the same day to avoid batch effect.

To obtain plasma, whole blood samples were centrifuged at 1650g for 25 minutes at room temperature. Subsequently, plasma samples from the 3 time points were bio-banked at −80 °C until study completion. Assays for each biomarker were run on the same day after study completion to avoid batch effect. We employed commercially available enzyme-linked immunosorbent assay (ELISA) kits to measure the level of 9 plasma biomarkers, namely IL-6, IL-1β (ThermoFisher, Waltham, MA), CXCL12/SDF-1α, CXCL5/RANTES (R&D Systems Inc., Minneapolis, MN), BDNF (Promega Corporation, Madison, WI), DHEA (CUSABIO, Houston, TX), cortisol, hs-CRP (Tecan, Männedorf, Switzerland), and sgp130 (RayBiotech Inc., Norcross, GA).

100 ng recombinant human (rh)CXCL12 (CXCL12/SDF-1α, R&D Systems Inc.) was mixed into 200 μL agarose and pipetted onto the wall of a 24-well plate culture well while the plate was tilted at a 45° angle to prevent the agarose from spreading across the bottom of the well before fully solidifying. The resulting gel body consists of a small ‘bead’ of approximately 3 mm in length at the edge of each well.
To test the protein release rate, BSA-conjugated fluorescein isothiocyanate (BSA-FITC, Sigma-Aldrich) was used as a model. Using the method previously stated above, 1 mg of BSA-FITC was mixed with agarose and added to 24-well plate culture wells. Release of the BSA-FITC was conducted by adding 1 mL of PBS to each well, and after each time point a 20 uL aliquot of the release solution (PBS containing released BSA-FITC) was removed and added to 180 uL of pure-PBS in a 96-well plate. After 24 hours, all aliquots taken and contained in the 96-well plate were examined using a fluorescent 96-well plate spectrometer (Spectra Max 190 Absorbance UV–VIS plate reader) at an excitation wavelength of 485 nm and the fluorescent intensity measured at the emission wavelength of 535 nm. Using a serial dilution in PBS, a calibration curve was then used to convert the data to mg/mL concentration and plotted relative to time (Additional file 1).