Recombinant mouse il 34

C57BL/6j mice were anesthetized by isoflurane inhalation. Fifty nanograms (Gomez-Nicola et al., 2013 (link)) of recombinant mouse IL-34 or recombinant mouse CSF1 (Biolegend, Nordic BioSite ApS, Copenhagen, Denmark) in 10 microliters of sterile PBS were injected in the cisterna magna. Analgesia was performed as described above. This treatment was performed either once or every 24 h for 3 consecutive days and the mice were sacrificed one day after the last injection. In the EAE experiments, three injections of cytokines were performed, starting at the onset of the disease (grade 2, loss of tail tonus). For each mouse, the day of first injection is referred to as day 0 (D0).

The isolated tubular cells were cultured (2 × 10⁵ cells/well/500 µL) in methylcellulose (R&D Systems, Minneapolis, MN, USA) (42%) as a three-dimensional (3D) culture and in the presence of FCS (25%) and RPMI (33%) according to our previous studies [26 (link),27 (link),33 (link)]. Those cultures were growing in the absence (CT) or presence of recombinant IL-34 (recombinant mouse IL34, BioLegend; final dilution 1–10,000 pg/mL). The cells were incubated for four weeks in a CO₂ incubator at 37 °C. Every 10–14 days, 50 µL/well of fresh media containing the relevant concentration of IL-34 were added to the cell cultures. At the end of the incubation period in MCS, 0.5 mL of PBS was added to the culture wells that contained 0.5 mL MC, mixed by pipetting, and collected from the suspension in 15-mL tubes. The tubes were centrifuged at 1600 rpm for 10 min. Most of the volume was removed and the remainder of around 100 µL was collected from the bottom of the tubes. This volume, which contained the cells, was smeared on a slide for histological examination and/or collected and kept at −70 °C in a lysis solution to be used for RNA extraction.