Optiprep

Hundred milliliter of tetracycline-induced cell culture was centrifuged at 4 °C, 8000 rpm, for 15 min. The cell pellet was either stored at −20 °C until use or immediately resuspended in 10 mL of ice-cold lysis buffer [PBS buffer, 0.6 % (v/v) Tween-20]. Cells were sonicated and the suspension was clarified by centrifugation at 4 °C, 8000 rpm, for 35 min.; the supernatant was left for 16–24 h at RT for particle formation. Subsequently, the lysate was layered on an OptiPrep (Sigma-Aldrich) gradient formed in ultra-clear tubes [2 mL of 30 % (v/v) OptiPrep, 2 mL 24 % (v/v) OptiPrep, 1.5 mL 18 % (v/v) OptiPrep, 1.5 mL 12 % (v/v) OptiPrep, and 1.5 mL 6 % (v/v) OptiPrep in ultra-clear water] and ultracentrifuged at 27,000 rpm for 16 h at 4 °C. Then, 500 µL fractions were harvested and analyzed by western blot using anti-HBsAg rabbit polyclonal antibodies (Abcam). Purity of the fractions was analyzed by SDS-PAGE with Coomassie R-250 staining. sHBsAg positive fractions of the highest purity were pooled, and OptiPrep buffer was replaced with PBS using Amicon Ultra 100 K centrifugal units (Merck Millipore). The thus prepared samples were used for further analysis.

Fifty milliliters of the tetracycline-treated cell culture was centrifuged at 4°C at 8,000 rpm for 15 min. The cell pellet was immediately resuspended in 5 mL of ice-cold lysis buffer (PBS buffer, 0.6% [vol/vol] Tween 20). The cells were sonicated, and the suspension was clarified by centrifugation at 4°C at 8,000 rpm for 35 min. The supernatant was left for 16 to 24 h at room temperature (RT) to form particles. Subsequently, the lysate was layered onto an OptiPrep (Sigma-Aldrich) gradient formed in ultraclear tubes (2 mL of 30% [vol/vol] OptiPrep, 2 mL of 24% [vol/vol] OptiPrep, 1.5 mL of 18% [vol/vol] OptiPrep, 1.5 mL of 12% [vol/vol] OptiPrep, and 1.5 mL of 6% [vol/vol] OptiPrep in PBS) and ultracentrifuged at 27,000 rpm for 16 h at 4°C. Next, 500-μL fractions were harvested and analyzed by Western blotting using anti-sHBsAg rabbit polyclonal antibodies (pAbs) (OriGene). The purity of the fractions was analyzed by SDS-PAGE with Coomassie R-250 staining. The fractions with the highest numbers of particles were pooled, and the protein concentration was measured by the Bradford assay (Bio-Rad). Finally, the OptiPrep solution was replaced with PBS using Amicon Ultra 100 kDa MWCO centrifugal units (Merck Millipore). These samples were used for further analysis and immunization.

Microglia were isolated from neonatal rat cerebral cortex as described previously (Zhuravleva et al., 2015 ). Briefly, pups were anesthetized and perfused via the left ventricle with cold DPBS solution supplemented with 5 U/ml sodium heparin. The cortex was isolated, and the pia mater peeled off. Tissue was mechanically homogenized and incubated with an enzyme solution (papain 2 mg/ml, DNAse 50 U/ml, dispase 2.5 U/ml) in DPBS for 30 min at 37°C with shaking at 180 rpm. Cells were separated using a 40 μm cell strainer followed by Optiprep (Sigma) discontinuous density gradient for 40 min at 300 g. Layers were cell suspensions in 19% Optiprep, 9% Optiprep, 7% Optiprep, DPBS. A microglia fraction was obtained from a layer, located between fractions 19 and 9%. The microglia were cultured in DMEM/F12 medium supplemented with granulocyte colony-stimulating factor (G-CSF,5 ng/ml, Sigma, United States), 10% FBS, 2 mM L-Glutamine and Penicillin-Streptomycin (PanEco, Russia) in humidified 5% CO2. Resulting cells were transplanted into the area of SCI after 24 h of culture.