Crl 2768

P0-expressing RT4 D6P2T myelinating Schwannoma cells (ATCC® CRL-2768™; [35 (link)]) and non-P0-expressing MDAMB 468 human breast cancer cells (ATCC® HTB-132™) were grown in Dulbecco’s modified Eagle medium (Life Technologies, UK) containing penicillin, streptomycin and foetal calf serum (All BD Biosciences) at 37 °C and 5% CO₂.
In line with their use by Whitney et al. [27 (link)], transgenic B6.Cg-Tg(Thy1-YFP)-16Jrs/J (THY-1 YFP) mice were obtained from JAX (the Jackson Laboratory) and were used for ex vivo and in vivo nerve staining and in vivo biodistribution studies (8–15 weeks old). THY-1 YFP mice express spectral variants of GFP (yellow fluorescent protein—YFP; ex 488, em 520) at high levels in motor and sensory neurons. The fluorescent signal in the nerves was used as an internal control for the staining (pattern) of the developed imaging agents. Balb/c nude mice were used as non-fluorescent control.

RT4-D6P2T cell line was provided by ATCC (CRL-2768, batch 3993689, mycoplasma-free). Cells were thawed and cultured at Neurofit facilities (Illkirch, France) in DMEM medium (ATCC) containing 10% Foetal Bovine Serum (FBS; ATCC) and 1% antibiotic-antimicotic mixture (Gibco), and maintained in a humidified incubator at 37°C in 5% CO₂-95% air atmosphere. Two days later, cells were trypsinised and transferred to 12-well plates (Nunc) at 30,000 cells per well. After 48 h, culture medium was replaced by DMEM containing drugs without FBS. After 8 h of treatment, cells were washed and harvested in 100 μL Phosphate Buffer Saline (PBS), then centrifuged 10 min at 13,000 rpm (4°C). The supernatant was discarded and the pellet was stored at −80°C until use. Three experiments with two independent cultures of RT4 were performed and each condition was done in triplicate.

The P0 specificity of Cy5-P0_101–125 was previously determined in P0-expressing myelinating Schwannoma cells (RT4 D6P2T myelinating Schwannoma cells (ATCC^® CRL-2768™; [34 (link)])) and non-P0-expressing cells, using P0 antibodies and a fluorescent derivative of the extracellular portion of P0 [27 (link)]. P0-expressing RT4 D6P2T cells were grown in Dulbecco’s modified Eagle medium (Life Technologies, Paisley, UK) containing penicillin, streptomycin, and fetal calf serum (All BD Biosciences) at 37 °C and 5% CO2. Cells were trypsinized and seeded onto 35 mm culture dishes that contained a glass insert (MatTek co) one or two days prior to the imaging experiment. One hour prior to imaging, 1 μM Cy5-P_101–125, Cy5-P0_101–120, Cy5-P0_101–115, Cy5-P0_101–110, Cy5-P0_101–106, Cy5-P0_105–125, Cy5-P0_108–125, Cy5-P0_112–125, Cy5-P0_116–125, Cy5-P0_120–125, or the original lead compound Cy5-P0_101–125 [27 (link)] was added (incubation at 4 °C; N = 3 per tracer). Imaging was performed as previously described [35 (link)]. Image acquisition and processing were performed using LASX software (Leica Application Software Suite 4.8). Image analysis, including (3D) surface plot and co-localization analysis, was performed using Image J [26 (link)].