Luciferin potassium salt

Mouse lumbar (L1–L5) DRG tissues were dissected with fine spring scissors and kept in chilled Hanks’ buffered salt solution (Invitrogen). Circadian bioluminescence measurement was performed as previously described^{21 (link)}. Briefly, all dissected tissues were cultured on Millicell culture membranes (PICMORG50, Millipore) and were placed in 35 mm tissue culture dishes containing 2 mL DMEM media (Invitrogen) supplemented with 352.5 μg/ml sodium bicarbonate, 10 mM HEPES (Invitrogen), 2 mM l-Glutamine, 2% B-27 Serum-free supplement (Invitrogen), 25 units/ml penicillin, 25 μg/ml streptomycin (Invitrogen), and 0.1 mM luciferin potassium salt (L-8240, Biosynth AG). Sealed dishes were placed in a LumiCycle luminometer (Actimetrics, Wilmette, IL) and bioluminescence was recorded continuously. For the paclitaxel treatment group, 20 µM final concentration paclitaxel recording media or vehicle recording media were used to culture DRG. For data analysis, we used the LumiCycle data analysis program to calculate circadian period and amplitude (Actimetrics, Wilmette, IL).

OAPs harbored a rapidly degradable form of luciferase, dLuc, driven by the Per2 gene promoter through lentiviral infection (Per2-dLuc)^{63 (link)} purchased from DNA/RNA Delivery Core, Northwestern University, using polybrene Infection Reagent (Millipore, #TR1003G). OAPs were maintained in a culture medium previously described^{60 (link)}, supplemented with 2 μg/ml blasticidin (Sigma-Aldrich, #15205) to select for stable Per2-dLuc integration. Selected cells were then trypsinized and seeded in 3.5 cm dishes (1 × 10⁵cells/cm² for immediate luciferase assay the following day or 8 × 10⁴ OAPs/cm² for siRNA or plasmid transfections prior to luciferase assay). The OAPs have been transfected before synchronization or received a pharmacological treatment after synchronization (described above). Cells were synchronized with 1 μM dexamethasone (Sigma-Aldrich, #D-1756) for 1 h. Cells were washed twice with PBS and the medium was replaced with 1.2 ml phenol-red free medium supplemented with 352.5 μg/ml sodium bicarbonate, 10 mM HEPES, 2 mM l-Glutamine, 5% FBS, 25 units/ml penicillin, 25 μg/ml streptomycin (all products from Life Technologies, ThermoFisher Scientific), and 0.1 mM luciferin potassium salt (Biosynth AG, #L-8240). Sealed cultures (VWR, cover glass #631-0177) were placed in a luminometer (Atto, AB-2550 Kronos Dio) at 37 °C.