Sodium deoxycholate

After an overnight incubation under anaerobic
conditions, OMVs were isolated as described
previously (23 (link)). Briefly, 500 mL of the bacterial
cultures were centrifuged at 6000 g at 4˚C. The
cell pellets were washed twice in phosphatebuffered solution (PBS). Then, the cell pellets were
resuspended in a 9% sodium chloride solution. The
cell suspensions were homogenized and concentrated
by centrifugation at 2900 g for 1 hour at 4˚C. The
total wet weight of cell pellets was calculated and
resuspended in 7.5 times the wet weight of 0.1 M
tris-10 mM ethylenediaminetetraacetic acid (EDTA)
buffer (Sigma-Aldrich, USA). The vesicles were
extracted by the addition of 1/20^th the volume of 0.1
M Tris, 10 mM EDTA, and sodium deoxycholate
(100 g/L) buffer (Merck, Germany). OMVs were
separated from cell debris at 20 000 g for 60 minutes
at 4˚C. The supernatant that contained the vesicles
was centrifuged at 20 000 g for 120 minutes at 4˚C in
order to concentrate the vesicles. The pellet was resuspended in 10 mM EDTA, 0.1 M Tris, and sodium
deoxycholate (5 g/L) buffer, and the suspension was
centrifuged again at 20000 g for 120 minutes at 4˚C.
The concentrated OMVs were resuspended in a 3%
sucrose solution. Finally, the suspension was filtered
through a 0.22-µm polyvinylidene difluoride filter
(Millipore, Billerica, MA, USA).

Trizma^® hydrochloride (Tris) (Merck, Dorset, U.K.) (10812846001), Sodium chloride (NaCl) (Merck, Dorset, U.K.) (71383), DPBS (Merck, Dorset, U.K.) (D8537), Sodium deoxycholate (Merck, Dorset, U.K.) (D6750), Triton 100 (Merck, Dorset, U.K.) (×100), Protease inhibitor cocktail (Merck, Dorset, U.K.) (P8340).

Ficoll, MEM α, L-glutamine, and Penicillin–Streptomycin were obtained from Sigma (St. Louis, MO, USA). FBS, PBS, and trypsin-EDTA solutions were purchased from Gibco Laboratories (Gaithersburg, MD, USA). EasySep was obtained from StemCell Technologies (Vancouver, Canada), rh M-CSF, and rh RANKL were purchased from PeproTech (London, UK).
Acetonitrile, water, formic acid, ammonium-bicarbonate, and sodium deoxycholate were obtained from Merck (Darmstadt, Germany). Methanol was purchased from VWR International (Debrecen, Hungary). Dithiothreitol, iodoacetamide, and TFA were purchased from Thermo Scientific (Waltham, MA, USA), and RapiGest SF surfactant was obtained from Waters (Milford, MA, USA). Trypsin/Lys-C Mix and Trypsin Gold were purchased from Promega Corporation (Madison​, WI, USA). All chemicals, reagents, and solvents were HPLC-MS grade.

Cells were harvested and lysed in RIPA buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100 (Merck), 0.5% (w/v) sodium deoxycholate (Merck), 0.1% (v/v) SDS, 5 mM DTT, 0.5 mM PMSF, 5 mM NaF and protease inhibitors (complete EDTA-free, Roche). Lysates were cleared by centrifugation and protein concentration quantified with Bradford. Equal amounts of total protein were loaded onto 4–12% gradient NuPAGE Bis–Tris gels (Invitrogen). Proteins were blotted onto 0.45 μm nitrocellulose membrane using wet transfers. Vinculin is the loading control.

Leupeptin, aprotinin, Pefabloc^® SC, Tween^® 20, Triton^® X-100, C.I-2 (calpain inhibitor 2), glycine and neutral red solution all the salts were purchased from Sigma-Aldrich. ECL ADVANCE® Detection System and Protein G-Sepharose were obtained from GE Healthcare. t-BOC (t-butoxycarbonyl)-Leu-Met-CMAC (7-amino-4-chlorometylcoumarin), a fluorogenic calpain substrate was purchased from Molecular Probes (Invitrogen). Sodium deoxycholate was from Merck (Milan, Italy). Anti-NMDAR2B monoclonal antibody (13/NMDAR2B) and anti-HSP90 monoclonal antibody (68/HSP90) were purchased from BD Transduction Laboratories TM. Anti-NR1, CT monoclonal antibody and anti-synaptophysin polyclonal antibody were purchased from Millipore. Monoclonal anti-calpain 1 (calpain I, subunit p80) clone 15C10 and monoclonal anti-calpain 2 (Domain III/IV) clone 107–82 were obtained from Sigma-Aldrich. Calpastatin was detected with the monoclonal anti-calpastatin (Domain IV) clone 1F7E3D10 purchased from Calbiochem. Anti-syntaxin monoclonal antibody (4H256) was purchased from GeneTex. Anti-PSD95 polyclonal antibody was obtained from Cell Signaling.

Amikacin disulfate was purchased from Alfa Aesar (Milan, Italy), and sodium deoxycholate, glycine, tris(hydroxymethil)aminomethane (TRIS) and 1-fluoro-2,4-dinitrobenzene (FDNB) were obtained from Merck Life Science (Milan, Italy). Water was purified by reverse osmosis. All organic solvents and the eluent components for the HPLC study were of analytical grade and purchased from Merck Life Science (Milan, Italy).