Topcount nxt counter

The TF-1 cell line (ECACC 93022307; Sigma-Aldrich, St. Louis, MO) was maintained between 0.5 and 9 × 100,000 cells/mL using RPMI 1640 medium supplemented with GlutaMAX (Invitrogen, Carlsbad, CA, USA), 10 % fetal bovine serum (Invitrogen), 1 % sodium pyruvate (Invitrogen), 1 % penicillin/streptomycin (Invitrogen) and 3 ng/mL human granulocyte-macrophage colony-stimulating factor (GM-CSF; eBiosciences, San Diego, CA, USA).
Cell suspensions were centrifuged, resuspended in culture medium without GM-CSF and seeded at a density of 12,500 cells/well in a 96-well plate. Cells were subsequently incubated for two hours with a dilution series of ALX-0061 in the absence or presence of 1 mg/mL recombinant HSA, after which 2 ng/mL recombinant hIL-6 was added and cells were further incubated for 72 hours at 37 °C in a humid chamber under a 5 % CO₂ atmosphere. During the last six hours of the incubation, cells were pulse-labeled with 0.2 μCi/well of ³H-thymidine (GE Healthcare). Cells were then harvested and the ³H-thymidine incorporation was measured using a Topcount NXT counter (PerkinElmer, Waltham, MA, USA). Results were expressed as average counts per minute per well.

EAO cells were transiently transfected using FuGene HD (Promega) transfection reagent according to the manufacturer’s instructions. One day before transfection, 2 × 10⁴ cells per well were seeded in a white 96-well plate (Nunc by Thermo Fisher Scientific, Roskilde, Denmark) and incubated at 27 °C for 24 h. Each well was transfected independently with a 4:1 ratio of reagent:DNA with 100 ng plasmid DNA. The cells were transfected with the reporter construct zfPer1b-Luc^{29 (link)} or D-box_Cry1a-Luc based on^{31 (link)}. As a control, zebrafish PAC-2 cells^{30 (link)} were transiently transfected in the same 96-well plate. Non-transfected cells were used as negative control for each cell line. After incubation for 24 h, the transfection solution was replaced by culture medium supplemented with 2 mM D-Luciferin (Biosynth, Bratislava, Slovakia). Cells were exposed to four 12:12 h LD cycles followed by 24 h DD. The bioluminescence was measured with a Topcount NXT counter (PerkinElmer, Shelton, USA) as previously described^{29 (link)}. We imported the data into Excel (Microsoft) by using the "Import and Analysis" macro (S. Kay, Scripps Research Institute). Mean and standard deviation of eight independently transfected wells were calculated with R 3.6.1⁵⁹ and shown using ggplot2⁶⁰ .

We used PAC-2 light-responsive cells stably transfected with the zper1b:luc reporter^{26 (link)}. 96-well culture plates were seeded with approximately 30,000 cells per well and placed in a dark room. Different lighting conditions were applied by exposure to a time-controlled white light source (Supplementary Fig. S1). Approximately every 40 min, each plate was automatically moved into the counting chamber of a TopCount NXT counter (Perkin Elmer) and luminescence was measured for approximately 5 min (3 s per well). Forskolin (FOR), dibutyryl cAMP (DBC), epidermal growth factor (EGF), U0126, phorbol-12-myristate-13-acetate (PMA), and ro-318220 (RO) were used at concentrations indicated in Supplementary Table S1. An overview of the experiment design is shown in Fig. 1A.