Ecotransfect

The sequence of the luciferase‐cAMP binding site fusion protein from the pGloSensor‐20F vector (Promega) was amplified and ligated into a bicistronic pIRESneo vector (Clontech, Mountain View, CA, USA) to obtain the reporter gene plasmid. The pcDNA5/FRT/TO vector (Invitrogen) was used to express the CXCR4 receptor constructs (cDNA Resource Center, Bloomsburg University, Bloomsburg, PA, USA) 71. Flp‐In T‐REx 293 cells (HEK 293, Invitrogen) were first stably transfected with the reporter plasmid using the Flp‐In system (Invitrogen) and EcoTransfect (OZ Biosciences, Marseille, France). Stable clones were selected with 1 mg/ml geneticin. A suitable clone was then chosen as host cell line for stable overexpression of the CXCR4 construct using the Flp‐In system with 250 μg/ml hygromycin B for selection.

Ten thousand COS7 fibroblast cells were plated onto 13 mm coverslips. Twenty-four hours after plating, cells were transfected with 1 μg of plasmid DNA using Ecotransfect (Oz Biosciences) according to the manufacturer's instructions. Cells were fixed 48 h after transfection in 4% paraformaldehyde, 4% sucrose for 20 min, and then permeabilized in 0.1% Triton 1% BSA and stained with primary antibodies [mouse anti-FLAG (M2), 1:1000; rabbit anti-GFP, 1:500] in 1% BSA in PBS for 2 h at room temperature. After three washes in PBS, secondary antibodies (anti-mouse Alexa Fluor 564 and anti-rabbit Alexa Fluor 488; Invitrogen) were applied at 1:500 in 1% BSA in PBS for 1 h in the dark. Coverslips were mounted on slides using Mowial mountant (10% Mowial, 25% glycerol in 0.1 m Tris, pH 8.5) containing 1 μg/ml DAPI. Images were acquired using a 40× objective on a Nikon TE200 epifluorescence inverted microscope using a RoleraXR CCD (QImaging) camera controlled by SimplePCI Software (Hamamatsu). The percentage of COS7 cells bearing neurite-like processes was measured. Processes were defined as being longer than the cell-soma diameter and having a width of <2 μm.

COS7 cells were plated at a density of 3 × 10⁴ cells per well of a 24 well plate or 10⁵ cells per well of a 6 well plate and transfected using EcoTransfect 24 h after plating according to the manufacturer’s instructions (Oz Biosciences, Marseille, France). Rat cerebellar granule and hippocampal neurons were prepared as previously described40 (link)41 (link) and approved by the Biology Ethics Committee, University of York. The animals were euthanised according to Schedule 1 of the UK Home Office Animals (Scientific Procedures) Act. Neurons were plated at a density of 2.5 × 10⁵ cells per coverslip and transfected after 24 h using a calcium phosphate kit (Promega, Southampton, UK) or Lipofectamine2000 (Invitrogen, Paisley UK) according to the manufacturer’s instructions. Following transfection the medium was replaced with MEM culture medium with 10 μM arabinofuranosyl cytidine for the indicated time before fixing for staining and morphological analysis. For L1-CAM assays, a 10 μl droplet containing 25 μg/ml L1-CAM extracellular domain (R&D Systems) and 1 mg/ml fluorescein isothiocyanate (FITC) was spotted onto the centre of a poly-D-lysine coated coverslip and air dried for 1 h prior to plating neurons.