Low bind tube

Cells were sorted in a flow cytometer (FACSVantage) equipped with a 100 μm nozzle, and operated using the FACSDiva software (BD Biosciences). Distinct populations of cells were isolated based on forward scattering, lateral scattering, and the intensity of EGFP or DsRed fluorescence. Sorted cells were collected into Eppendorf® low-bind tubes with 100 μl HBSS without CaCl₂ and MgCl₂, with 10 mM HEPES, 1 mM EDTA, and 0.1% BSA. The Tau-EGFP negative fraction consisting of SGNNCs was further cultured for transfection with neurogenic factors or an empty vector. After culturing for 6–10 days, we collected induced neurons from the Tau-EGFP and DsRed double positive fraction. We also collected the Tau-GFP negative DsRed positive fraction transfected with an empty vector as a negative control. Lastly, we collected Tau-EGFP positive endogenous PANs at P1 to profile these cells and compare them to iNs.

Total RNA extraction was performed using 200 µl cCM or 200 µL pooled sEV fractions (fraction 8–11) together with the miRNeasy mini kit (Qiagen, Hilden, Germany). Synthetic oligonucleotides obtained from the miRCURY Spike-In kit (Qiagen) were added to the Qiazollysis buffer (Qiagen) before homogenization of the cCM/sEV samples. Glycogen (5 mg/mL) was added to the chloroform extract at 1:100 dilution to enhance precipitation. All other steps were performed according to the recommendations of the manufacturer. Total RNA was eluted in 30 µL nuclease-free water and stored at −80 °C in low-bind tubes (Eppendorf, Hamburg, Germany) until further analysis.

Preparation and analysis using LCM and mass spectrometry (MS) was done as previously described [14 (link)]. Tissue sections of FFPE human duodenal biopsies or mouse small intestine (5 μm) were adhered to PEN-covered slides (Zeiss) and dried at 37°C. The dry sections were dewaxed in xylene and rehydrated using ethanol and water. Visualization of cells was done using Mayer’s haematoxylin solution (Sigma). A PALM MicroBeam laser capture microdissection system (Carl Zeiss MicroImaging, Munich, Germany) was used to collect the isolated tissue into 0.5 mL opaque adhesive cap tubes (Zeiss). For all samples about 350 000 μm² of tissue was collected from either the apical or lateral part of the epithelial cell layer.
Dissected tissue was retrieved from the adhesive caps using 20 μl 0.1% ProteaseMax surfactant (Promega, WI, US) in 50 mM ammonium bicarbonate (ABC) and transferred to Low-Bind tubes (Eppendorf, Germany). The samples were heated for 90 min at 98°C followed by 1 h sonication in a water bath. Reduction was done by adding 2 μl 100 mM dithiothreitol and incubation at 56°C for 20 min before adding 2 μl 55 mM iodoacetamide for alkylation and incubating in 24°C for 30 min in the dark. Digestion was done by addition of 1.5 μl 0.01 μg/μl trypsin (Promega) with incubation overnight in 37°C and ended by acidification of the samples.

Confluent monolayers in 10-cm dishes were rinsed in PBS and lysed (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, and complete protease inhibitor). Lysates were transferred to low-bind tubes (Eppendorf) and incubated for 30 min at 4°C. Cell debris was pelleted by low-speed centrifugation (500g, 2 min). Cytosolic protein extracts were adjusted to comparable protein concentrations and incubated with indicated antibodies overnight at 4°C, on a rotating wheel. Dynabeads (Life Technologies) were added and samples were rotated for 1 h at 4°C. Tubes were placed in a magnetic rack (Life Technologies), and captured protein complexes were rinsed five times in lysis buffer. Beads were resuspended in sample buffer, and proteins were eluted by boiling for 5 min at 95°C. Bead-free samples were transferred to new reaction tubes. For isolation of proteins bound to NM-GFP, GFP-trap magnetic beads (ChromoTek) were used according to the manufacturer’s instructions.

Phalloidin staining of muscle fibres and scoring of muscle defects were performed as described previously^{65 (link)}. Briefly, synchronized L1 larvae were grown to the young adult stage (before the appearance of eggs), and collected and washed with M9 buffer. The worms were then transferred to 1.5 ml low-bind tubes (Eppendorf) and fixed with 4% formaldehyde (Thermo Fischer) in 0.1 M Na₂HPO4 for 15 min. The worms were pelleted, permeabilized in 500 μl of pre-chilled acetone (5 min at −20 °C) and then washed three times with PBS-TG (PBS, 0.5% Triton X-100 and 30 mM glycine). Rhodamine phalloidin (500 μl; R415, Life Technologies; 1:250 in PBS-TG) was added to the worms for 30 min at room temperature with rotation. The worms were washed three times, settled on poly-l-lysine-coated slides and mounted with ProLong Gold (P36930, Thermo Fischer).