P4957

In order to achieve the PLO coating on glass coverslip, first it was cleaned with ethanol and acetone by sonication. The cleaned glass coverslip was incubated in 30% PLO commercial stock (Sigma P4957, in PBS) at 37 °C in a 5% CO₂ incubator overnight. After the incubation, the coverslip was washed with sterile water and was dried and sterilized under UV for 20 min. The punched PDMS replica was cleaned with water followed by ethanol under sonication and then air-dried. It was incubated in DMEM/F-12 and Neurobasal medium (1:1) supplemented with 100 μg/ml penicillin-streptomycin (Biochrom) and 2 mM l-gluta-mine (Biochrom) (PSG), N2 and B27 supplement (N2B27, Thermo Fisher) at 37 °C in a 5% CO₂ incubator for two days. After the incubation, the PDMS replicas were washed under sonication with sterile water and dried and sterilized under UV for 20 min. The dried PDMS replica was placed and gently pressed on the PLO-coated glass cover-slip. Subsequently, Laminin (5 μg/ml, LN521, biolamina) (PLO-Laminin), was applied inside the chambers. The prepared assembly was placed under desiccation to remove air bubbles from the micro-channels. Afterward, the assembly was incubated at 37 °C for 2 h or at room temperature overnight.

On day 10 after the first injection, PBS- and cisplatin-treated WT mice were euthanized with C0₂ and perfused with ice-cold PBS. DRGs were quickly and carefully removed and trimmed in ice-cold DMEM, digested with Trypsin (0.3 mg/ml, Worthington, #LS003702) and collagenase D (1.5 mg/ml, Sigma, #11088858001) for 40 min at 34°C. Debris was removed by two successive centrifugations (6 minutes at 600 rpm) and cells were plated onto coverslips coated with 0.01% poly-L-ornithine (Sigma, P4957) and incubated overnight at 37°C with 5% CO2 in serum free DMEM as previously described [4 (link)].