Cytospin 4 centrifuge

Nuclei were isolated from iPSNs and postmortem human motor and occipital cortex tissue using the Nuclei Pure Prep Nuclei Isolation Kit (Sigma Aldrich) following manufacturer protocol with slight modifications as previously described [4 , 5 (link)]. About 10 million iPSNs or 100 mg of frozen postmortem motor cortex tissue (obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 2: Table 2 for demographic information) was used for nuclei isolation. A 1.85 M sucrose gradient was used to enrich for neuronal nuclei. Following isolation, nuclei were centrifuged onto collagen coated (1 mg/mL; Advanced Biomatrix) slides with a CytoSpin 4 centrifuge (Thermo Fisher Scientific) and immunostained as previously described [4 , 5 (link)] (see Additional file 2: Table 3 for antibody information). Isolated nuclei were subsequently imaged by super resolution structured illumination microscopy (SIM) using a Zeiss ELYRA S1 as previously described [4 , 5 (link)]. All images were acquired using identical imaging parameters (e.g. laser power, gain) and subjected to default SIM deconvolution and processing in Zeiss Zen Black 2.3 SP1. Representative images are presented as 3D maximum intensity projections generated in Zeiss Zen Black 2.3 SP1. Images were faux colored green for contrast and display.

BALF collection was performed with the method of Shin et al., (2015) (link). Briefly, to obtain BALF, ice-cold PBS (0.7 ml) was infused into the lungs two times and withdrawn each time with a tracheal cannula (total volume, 1.4 ml). The collected BALF was centrifuged at 1,000 rpm for 10 min at 4°C. The supernatants were collected and stored at −70°C before cytokine analysis. The cell pellet was re-suspended with 500 μL ice-cold PBS and attached on a slide using a Cytospin 4 centrifuge (Thermo Scientific, Waltham, MA, USA) (1,000 rpm, 5 min, 20°C). Differential cell count was performed with Diff-Quik® staining reagent according to the manufacturer’s instructions. Five images of each slide were captured with a Leica DM5000B microscope and the Leica Application Suite acquisition software (Leica Microsystems, Wetzlar, Germany) under 40× objective lens. Thereafter, total cells, eosinophil, macrophages, and other cells (neutrophils and lymphocytes) were counted. The levels of IL-4, IL-5, IL-6, IL-13, eotaxin (R&D system), and MUC5AC (Cusabio Biotech Co.) in BALF supernatant were measured using ELISA kits, according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader (iMark^TM, Bio-Rad Laboratories, Richmond, CA, United States).

Cells collected on day 11 were cytospun onto glass slides using a Cytospin 4 centrifuge (Thermo Scientific, Springfield, NJ), stained with Wright-Giemsa (Fisher Scientific, New York, NY), and observed with a Leica inverted contrasting microscope fitted with a camera (DFC420, Leica Camera, Inc, Allendale, NJ)

Cells were deposited on poly-lysine coated slides (J2800AMNZ, Thermo Scientific) using a Cytospin 4 centrifuge (Thermo Scientific) at 600 rpm for 10 minutes. Soluble cell fraction was pre-extracted by incubation with cold cytoskeleton buffer (CSK: 10 mM PIPES, pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.7% Triton X-100) (2 x 3 minutes), fixed with 4% PFA-PBS, and saturated with 3% BSA-PBS for 1h at RT. Anti-BLM antibodies (ab476, Abcam; sc-365753, Santa Cruz) were diluted at 1:200 and anti-nucleolin (ab22758, Abcam) was diluted at 1:1000 in saturation buffer and incubated on slides in a humid chamber for 90 minutes. Slides were washed 3 x 5 minutes with PBS-0.01% Tween, incubated protected from light in a humid chamber with secondary antibody (A11008, Invitrogen) 1:500 for 45 minutes at RT. Washed again 3 x 5 minutes with PBS-0.01% Tween, incubated with DAPI (20 μg/ml) in H₂O for 5 minutes and washed 3 times with H₂O. Slides were air dried and mounted with Prolong Gold (P36930, Invitrogen) and let to dry overnight. Image acquisition was performed with a ZEISS Axio Imager Z1 Apotome microscope and analysis was done with Omero server.