Labtek permanox chamber slides

Primary motor neuron cultures were prepared as previously described^{62 (link)}. Motor neurons were isolated and plated in eight-well LabTek Permanox chamber slides (Nunc, Roskilde, Denmark) pre-coated with poly-L-Ornithine and laminin. Cells were cultured for 7–10 days in Neurobasal medium supplemented with 2% B27, 2% (v/v) horse serum, glutamine 100 U/ml penicillin, and 100 μg/ml streptomycin. To induce cellular death, cells were treated with H₂O₂ (60 µM; Sigma-Aldrich) alone or together with 10 µl of empty MSPs for 48 h. For each individual experiment, a well with unstimulated cells were used as a positive control. Supernatant was collected, centrifuged at 12.000×g, and the level of glutamate was estimated by Glutamate Assay Kit (Sigma). Cells were fixed with 4% formaldehyde and nuclear morphology was visualized with Hoechst 33258 (1:1000, Invitrogen). Random images were recorded using Zeiss Axiovert 100 microscope mounted with an AxioCam MRm camera (Zeiss) and cellular survival was assessed as previously described^{44 (link)}.

THP-1 cells were differentiated on LabTek permanox chamber slides (Nunc, Thermo fisher scientific, Waltham, MA, USA) with 1µM PMA in full cell culture medium for 24 h at normal cell culture conditions (3.3×10⁵/well/300µl). After replacement with serum free medium, apoptotic Jurkat cells (1.65×10⁶ in 200µl serum free medium) and 50 nM PCI-Cy3 were added simultaneously and incubated for 2 h at RT. In control experiments untreated and CT Jurkat cells were incubated with PCI-Cy3 under the same conditions but in the absence of macrophages. After washing, cells were fixed in 4% PFA for 20 min, RT. Macrophages were stained with 25F9-FITC antibody (Santa Cruz, Dallas, TX, USA) and Jurkat cells with mouse anti human beta3 integrin antibody (1µg/ml) for 1 h at RT (Bioscience Research Reagents, Temecula, CA, USA) and secondary goat anti-mouse IgG Alexa488 coupled antibody (400 ng/ml) (Life Technologies, Carlsbad, CA, USA) for 45 min at RT and Hoechst (diluted 1∶1000) for 10 Minutes at RT. Fluorescence microscopy was performed on an Olympus AX70 microscope (Olympus, Tokyo, Japan), equipped with filter cubes for Hoechst: excitation 360 nm, emission 420–460 nm; FITC: excitation 450–480 nm, emission barrier filter 515; Cy3: excitation 525–560, emission 570–650 nm. Cell∧P software was used for acquisition.

Cells were seeded in 8-well Lab-tek Permanox Chamber slides (Nunc, Zellik, Belgium), treated for 24 hr, fixed in 4% paraformaldehyde for 15 min at room temperature, and ice-cold methanol for 5 min. Primary antibodies recognizing γH2AX (JBW301, Millipore, Overijse, Belgium) and RAD51 (PC130, Merck, Darmstadt, Germany) followed by secondary antibodies conjugated to Alexa Fluor 647 and 488 (Life Technologies) were used. Images were acquired using an A1R Eclipse Ti inverted confocal microscope (Nikon, Brussels, Belgium) and processed using Fiji software, with compound or vehicle-treated cells being processed identically. Nuclei with >5 foci were scored as positive, and at least 200 nuclei were counted per condition by two independent individuals, blinded to the genotypes.