X well tissue culture chamber

Mouse dorsal root ganglion (DRG) cultures were prepared from embryonic day 15 (E15) C57BL6 mouse embryos according to a previously published protocol [21 (link)]. DRG dissociation was performed in 0.05 % trypsin (Gibco) and 0.05 % collagenase (Sigma-Aldrich) for 20 min at 37 °C. Dissociated DRGs were resuspended in 40 μg/ml deoxyribonuclease I (Sigma-Aldrich), and the digestion was stopped with L15 Leibovitz’s medium (Gibco) containing 10 % horse serum (Gibco). For each condition approximately 500000 cells were seeded onto one collagen (Becton-Dickinson, Franklin Lakes, NJU, USA) coated x-well tissue culture chamber (Sarstedt, Nümbrecht, Germany) and incubated in growth medium at 10 % CO₂ for 4 to 5 days. Myelination was induced by changing to mouse myelination medium [21 (link)] supplemented with 20 mg/ml dialysed IVIG or dialysed control buffer. After 7 days of buffer/IVIG treatment, cultures were fixed with 4 % paraformaldehyde (PFA, Merck, Darmstadt, Germany) and stained against myelin basic protein (MBP) and β-tubulin (TUJ1). MBP positive internodes were counted in both conditions and the average number was determined.

BMDMs (10.000 cells per well) were grown, stimulated as indicated in X-well tissue culture chamber (#94.6150.401, Sarstedt, Germany), and further incubated for 48 h. BMDMs were washed with PBS and subsequently incubated for 10 min at 37 °C in 500 µl of 5 µM MitoSOX red reagent (#M36008D, Thermo Fisher Scientific, USA) protected from light. BMDMs were washed three times and one drop of ProLong GOLD Anti-fade Mountant with DAPI (#P36931, Thermo Fisher Scientific, USA) was added per well. The fluorescence imaging was performed with an Axiophot Zeiss microscope using a digital camera with AxioVision 4.8 software. The obtained images were analysed by Fiji ImageJ software.

For immunohistochemistry analyses cells were seeded on x-well Tissue Culture Chambers (Sarstedt, Nümbrecht, Germany) and transfected for 48 h. Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.2% Tween20 for 5 min in phosphate-buffered saline. Cells were stained for 1 h with the primary antibodies: anti-MiD51 (1:100) (Proteintech), anti-Tom20 (1:100) (Abcam, Cambridge, UK), anti-insulin (1:100) (Abcam) and anti-LC3 (1:100) (Sigma). Cy5 or FITC-coupled secondary antibodies (1:250) were used for visualization (Molecular Probes Invitrogen, Darmstadt, Germany). Cells were mounted and counterstained using Roti^®-Mount FluorCare DAPI (Roth, Karlsruhe, Germany) and analyzed using a Fluoview FV10i confocal microscope (Olympus, Hamburg, Germany).