Phorbol 12 myristate 13 acetate pma

Primary dissociated hippocampal cultures were established and transfected as described previously [23 (link)]. The cultures were transfected with the indicated plasmids at 14 days in vitro (DIV) and assays were performed 48 hours later at 16 DIV. Pre-warmed and CO₂-equilibrated Neurobasal media was used for all media replacements. For KCl stimulation experiments, media was removed and replaced with Neurobasal containing 30 mM KCl for 5 min, which was then replaced with unsupplemented Neurobasal for an additional 55 min. For PMA stimulation experiments, media was replaced with Neurobasal containing 100 nM Phorbol 12-myristate 13 acetate (PMA) (Enzo Life Sciences, Farmingdale, NY, USA) for 1 hour. For the assays with PKC inhibition, media was replaced with Neurobasal containing 1 μM GF 109203X for 30 min prior to KCl or PMA treatment as above, with 1 μM GF 109203X included in the stimulation media. Cells were fixed and processed as described for in situ hybridization and immunocytochemistry. Neurons having pyramidal-like morphologies were selected, and apical dendrites were chosen for analysis based on structural properties. Images were acquired on a Nikon Eclipse E800 microscope using PictureFrame software (Optronics, Muskogee, OK, USA).

Human whole blood was collected via venipuncture from healthy donors. The Institutional Review Board from the University of Toledo College of Medicine and Life Sciences approved the protocols, and written informed consent was obtained from all donors, in accordance with the Declaration of Helsinki. Blood was drawn into BD vacutainer tubes containing 12 mg K3 (tripotassium) EDTA and polymorphonuclear (PMN) cells were isolated by using a Polymorphprep™ gradient solution (Axis Shield) following manufacturer’s instructions. PMN cells (5.0 x 10⁷ cells/ml) in 0.2% BSA/HBSS++ buffer, were activated using 20 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Enzo Life Science) for 30 min at 37°C. The PMNs were centrifuged at 600 x g for 10 min. The supernatant was removed and centrifuged at 13,000 g for 2 min to remove cell debris. PMNs without PMA treatment were prepared as a control. The supernatants from PMA-activated and non-activated PMNs were assayed for properdin concentration, as indicated below.

CBD (C-045), Δ⁹-THC (T4764), LPS from Escherichia coli O111:B4 (L4391), and adenosine 5′-triphosphate (ATP) disodium salt hydrate (A6419) were obtained from Sigma Aldrich, Oakville, ON, Canada. Phorbol-12-myristate-13-acetate (PMA) was purchased from Enzo (BML-PE160-0005), Farmingdale, NY, USA. CBD and THC were provided as 1 mg/mL stock solutions in methanol and stored at −20 °C. Both cannabinoids were chosen to be used at a final concentration of 5 µM based on our optimization and previously published results [25 (link),26 (link)]. LPS was prepared by dissolving 1 mg powder in 1 mL of sterile phosphate buffer saline (PBS) to obtain 1 mg/mL solution as per the manufacturer’s instructions. PMA was dissolved in dimethyl sulfoxide (DMSO), cell culture reagent (CAS 67-68-5) [sc-358801, Santa Cruz Biotechnology (SCBT), Dallas, TX, USA] and further diluted to appropriate concentrations using sterile PBS. Trypan blue solution, 0.4% (15250061) was acquired from ThermoFisher Scientific (Life Technologies Inc., Burlington, ON, Canada).