Genios

Overnight LBS cultures of each strain were pelleted, washed once, and resuspended in 1 ml of the intended growth medium. Luminescence growth curves were performed in 20 ml SWT in flasks shaking at 225 rpm; periodic 1-ml and 300-μl aliquots were taken for luminescence and OD₆₀₀ readings on a luminometer (Turner Designs) and Tecan Genios plate reader, respectively. Growth curves were performed in 1.2 ml growth medium and monitored in the Tecan Genios plate reader with continuous shaking at high speed.

Estimation of DNA concentration and purity was performed using a spectrophotometer (BioSpectrometer^® basic, Eppendorf AG, Hamburg, Germany) at 260 and 280 nm. For a quantitative determination of DNA, the fluorescence signal was measured after addition of the DNA intercalation dye PicoGreen (Quant-iT™ PicoGreen^® dsDNA Reagent, Thermo Fisher Scientific Inc., Waltham, MA, USA) against a λ-DNA (Thermo Fisher Scientific Inc., Waltham, MA, USA) standard curve using a microtiter plate reader (GENios, TECAN Group Ltd., Männedorf, Switzerland).

Serum MOG_35–55-specific antibody was detected by enzyme-linked immunosorbent assay (ELISA). The 96-well microplates were pre-coated overnight with 10 µg/mL MOG_35–55 peptide at 4°C and blocked with 3% bovine serum albumin in PBS containing 0.1% Tween-20 (PBST) for 1 h. The plates were subsequently incubated with 100 µL mouse serum (1/100 dilution) at 37°C for 1 h. Plates were washed three times with PBST and the appropriate horseradish peroxidase-conjugated goat anti-mouse IgG was added to detect the bound Ig for an hour at 37°C. After washing, the plates were colorized with tetramethylbenzidine and absorbance was read at 450 nm. The cutoff value was defined as the mean optical density value of control samples plus two SDs.
Chemiluminescent enzyme-linked immunosorbent assay (CLISA) was used to detect MOG_35–55-specific antibody in cell culture supernatant because of the anticipated low titer of the antibody. This procedure was similar to conventional ELISA except for the substrate solution. After adding the Lumigen PS-atto substrate (Lumigen, Inc., Southfield, MI, USA), the chemiluminescence intensity was monitored using a luminescence reader (GENios, Tecan Group Ltd., Männedorf, Switzerland). The test for repeatability of this method was presented in Figure S4 in Supplementary Material.