All patients with FMS fulfilled the American College of Rheumatology’s 1990 criteria 4 and did not have comorbid ME/CFS. Serum from such patients was already in our possession from a previous study (Madrid, Spain). 29
The normal controls were not related to any of the ME/CFS or FMS patient groups and were purchased from BBI Solutions (Cardiff, UK). Serum samples were labeled only with a code number, the age and gender of the subjects. All samples were processed immediately, and the serum was stored in –80°C until used for analysis.
Total EVs Isolation: Total EVs were isolated using the exoEasy Maxi Kit (Qiagen, Valencia, CA) from 1 mL of serum. Pre-filtered serum (0.8 μm syringe filter) were mixed with Buffer XBP and were bounded to an exoEasy membrane affinity spin column. The bound EVs were washed with Buffer XWP, were eluted with 400 µl Buffer XE (an aqueous buffer containing primarily inorganic salts) and were then ready to use for further analysis. The reason we used this commercialy available EVs purification kit is due to the limited amount of biological samples (2 mL serum from each ME/CFS patient and healthy control) available in our possession.
BCA assay: The concentration of total protein was quantified by the bicinchoninic acid (BCA) assay (Thermo Fisher Inc., Rockford, IL) using bovine serum albumin (BSA) as standard.
Electron microscopy: A drop of isolated serum-derived total EVs suspended in Buffer XE was deposited on Formvar-carbon-coated electron microscopy grids, fixed as above, immunolabelled and stained using the method as described before. All samples were analyzed at Harvard Medical School’s Electron Microscopy (EM) Core Facility by Ms. Maria Ericsson, Manager of the Harvard Medical School EM Facility, using the Tecnai G2 Spirit BioTWIN transmission electron microscope (TEM).
Mitochondrial DNA: Total DNA was extracted from EVs using Qiagen DNA Micro extraction kit (Qiagen, CA). Mitochondrial specific DNA for 7S (mt-7S) was quantified by RT-qPCR using Taqman gene expression assays (Mt-7S: Hs02596861_s1; GAPDH: Hu, VIC, TAMRA, Applied Biosystems, Carlsbad, CA). Samples were run at 45 cycles using Applied Biosystems 7300 Real-Time PCR System. GAPDH DNA was used to exclude any genomic “contamination.”
Cell culture: SV40 immortalized human adult microglia, frozen in the M1 pro-inflammatory state, were purchased from Applied Biological Materials Inc., (ABM, Vancouver, Canada). Microglia were cultured in Prigrow III medium (ABM) supplemented with 10 % FBS and 100 U/mL penicillin/streptomycin using BD PureCoat ECM Mimetic Cultureware Collagen I peptide plates (Thermo Sci.).
Cell viability assay: Cell viability was measured by Trypan blue (1%) exclusion.
Mediator assay: IL-1b was assayed using commercial ELISA kits from R&D Systems (Minneapolis, MN). Control cells were treated with equal volume of culture medium only.
Power analysis and statistics: Primary objective: Content of serum EV-mtDNA from ME/CFS and FMS patients. The required sample size to observe a difference of 30% in EV-associated mtDNA between ME/CFS patients and controls at 5% significance level with a power of 80% is at least 25 subjects/group. Since the variance between the two subgroups with and without FMS is not known, the number was increased to n=30 per group. Correlations between mtDNA and either subgroup were determined using the Spearman rank correlation test. Comparisons with control were done using either parametric t-test for independent samples or Mann-Whitney non-parametric test depending on normality of distribution to be checked with the Shapiro–Wilk’s test. Comparisons between the groups were done with ANOVA and Wilcoxon post-hoc paired rank sum test.