The HC2S2 rat neural stem cell line that was described previously [12,13] was utilized for the isolation of exosomes. Cell culture petri-dishes (10 cm) were coated with Matrigel for 2 hours at room temperature prior to use. Cells were cultured in DMEM/F12 supplemented with N2 medium and 20 ng/ml basic fibroblast growth factor. Cells were grown at 37°C in 5% CO2 to 80 – 85% confluency before passage.
Heat-shock of HC2S2 cells
Once cells reached 80% confluency, media was collected and utilized for non-heat shock (NHS) exosome isolation. Then, fresh media was added, and cells were incubated at 42°C in 5% CO2 for three hours to stimulate HS conditions . Subsequently, cells were returned to 37°C in 5% CO2 for 21 hours, and media was then collected for isolation of exosomes from heat shock-derived cells.
Exosomes were isolated and purified using a sucrose gradient ultracentrifugation method adapted from our previously described protocols [15,16]. The cell culture media was collected by centrifugation at 300 x g for 20 minutes to remove cellular debris. The supernatant was centrifuged at 1,000 x g for 15 minutes to remove apoptotic bodies. The supernatant was then pooled and ultracentrifuged at 100,000 x g for 90 minutes in the Beckman SW41Ti Rotor using Beckman Coulter ultracentrifuge tubes. For fractionation, the supernatant was discarded, and the resulting pellets (crude exosomes) were resuspended in 0.32 M sucrose and added to the top of a sucrose gradient with the following concentrations of sucrose in 5 mM HEPES dissolved in dH2O with a pH of 7.4: 0.72 M, 1.16 M, 1.58 M and 2.0 M. The sucrose gradient was then ultracentrifuged at 100,000 x g for 16 hours. Next, ten 1 mL fractions were collected using a fractionation machine and each individual fraction was added to 1X PBS and further centrifuged at 100,000 x g for 60 minutes. The supernatant was discarded, and the pellets were resuspended in 1X PBS and stored at -80°C until use. All centrifugation steps were carried out at 4°C.
Nanoparticle Tracking Analysis (NTA) was conducted to assess the concentration and size of exosomes collected from HS- and NHS-derived cells. The NanoSight NS300 system utilized 100 µg of exosomal protein diluted in 1X PBS for comparison between the size and concentration of exosomes isolated from the two conditions (Malvern Instruments). Transmission Electron Microscopy (TEM) was conducted to further validate the morphology of the collected samples adapted from a previously described protocol . Briefly, NHS and HS-derived exosomes were resuspended in water containing 0.2% paraformaldehyde (PFA). Samples (10 microliters each) were prepared by drop-casting on a graphine-based grid and then 1% phosphotungstic acid was applied as a contrast agent. Samples were imaged using the FE-TECNAI-G2 transmission electron microscope (FEI) with an exposure of 1.0 sec and magnification of SA 59,000.
Western blot analysis was conducted on the 10 isolated fractions to determine the presence of known exosomal markers. Proteins from each fraction were separated onto 12% Bis-Tris gels and transferred to polyvinylidene difluoride membranes. Antibodies against HSP-90 and Flotillin-1 were used to characterize the isolated fractions.
Mass spectrometric analysis of exosomal proteins
The exosomal proteins were separated by SDS-PAGE, and the gel was stained with Coomassie G250. Each gel line corresponding to each replicate/group was cut into small slices before being subjected to destaining and in-gel digestion as previously described [18,19,16]. The tryptic peptides were analyzed in an Easy nLC 1200 using a trapping and desalting online trap column (300 μm x 20 mm Acclaim PepMap C18 100Å (Thermo Scientific), and were separated by an Acclaim PepMap RSLC 2 μm, 75 μm x 15 cm, nanoViper (Thermo Scientific) coupled to nanoESI QExactive Plus Orbitrap HR/MA mass spectrometer as previously reported [20,18]. The ions were analyzed in positive MS ion mode (m/z 300 −1800) with 70,000 resolution (m/z 200) after accumulation with target ions to 1 × 106 value based on predictive AGC. The MS/MS ions selection was set at m/z 65-1800 to 1 × 105 counts. The spectrum was deconvoluted and analyzed using the Mascot Distiller v2.6 (www.matrixscience.com) and Discovery and Proteome Discoverer v2.1 (Thermo Scientific). A mascot generic format list (MGF format) was generated to identify +1 or multiple charged precursor ions from the MS data file.
Mascot server v220.127.116.11 (www.matrix-science.com, UK) in MS/MS ion search mode (local licenses) was applied to conduct peptide matches (peptide masses and sequence tags) and protein searches against SwissProt 2021_04 (565,928 sequences; 204,173,280 residues) using taxonomy filter for Rattus norvegicus. The protein redundancy that appeared at the database under different GI and accession numbers was limited to Human. All the proteins identified in the current study were found in these domains.
Bioinformatic analysis of NHS and HS exosomal proteins
The DAVID functional annotation tool (https://david.ncifcrf.gov/summary.jsp) was utilized to perform gene ontology (GO) and pathway analysis of significant interactors. The UNIPROT IDs for each protein were converted to the official gene ID with Rattus norvegicus selected as the species. The default settings for thresholds of 2 genes per term and an EASE threshold of 0.1 were utilized for the analysis. The p-value was converted to the -log(p). The Venny 2.1 software (https://bioinfogp.cnb.csic.es/tools/venny/) was utilized to determine differences in GO terms and pathways between NHS and HS exosomal proteins.
Treatment of neuronal cultures with NHS- or HS-derived exosomes in the presence of oxidative stress or Aβ peptide
Mouse neuronal cells obtained from the Coriell Institute for Medical Research were cultured in 12-well plates in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics as previous described . The cells were co-treated with 250 µM H2O2 in the presence of 2 µg/ml of crude NHS- or HS-derived exosomes for 8 h. Alternatively, neuronal cultures were treated with 250 nM of Aβ1-42 peptide that we previously used  in the presence of 4 µg/ml of purified NHS or HS-exosomes (pooled fraction 3-8) for 24 h. Following the treatments, the cells were collected for assessment of cell viability by measuring ATP levels using an ATP assay kit as previously described .
All numerical data are presented as mean ± SD. For comparison between NHS- and HS-derived exosomes in terms of concentration and diameter, a student’s t test was utilized. All other analyses were carried out using a one-way ANOVA followed by a Tukey’s post hoc test. Statistical significance was accepted when p < 0.05. For statistical analysis and graphical displays, GraphPad Prism Statistical Software version 8.2.1 was utilized.