Mgso4

Total RNA (100 ng) was reverse transcribed using Super Script III Reverse Transcriptase (Thermo Fisher Scientific) with the DPB1-R primer. First PCR for the coding region of HLA-DPB1 locus was performed with the following reaction including 1 μl of the first-strand cDNA solution, 16.99 μl of Nuclease-Free water, 2.5 μl of 10× High Fidelity PCR Buffer, 2.2 μl of 2.5 mM dNTP, 1.2 μl of 50 mM MgSO4 (Thermo Fisher Scientific), 0.11 μl of 5 unit/μl Platinum Taq, 0.5 μl of 10 μM DPB1-L, 0.5 μl of 10 μM 5′ end biotinylated primer. The following cycling conditions were used: 1 cycle of 94 °C for 30 s, 30 cycles of 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 1 min. The second PCR was performed using the purified first PCR products and phosphorylated DPB1-L (P-DPB1-L) in 8 wells (total 160 μl). The cycling conditions were the same as those of the first PCR. Total PCR products were purified using AMPure XP. The primer sequences are shown in Fig. 5a.

Campylobacter jejuni was routinely grown on blood agar base No.2 plates (BA plates; Oxoid) supplemented with 5% horse blood (TCS Biosciences) under microaerobic conditions in either a modular atmosphere controlled cabinet (5% CO₂, 5% O₂, 2% H₂, 88% N₂) or in anaerobic jars using gas replacement (7.3% CO₂, 5.6% O₂, 3.6% H₂, 83.5% N₂) at 42 °C. Bacteriophage DA10 was isolated as described previously [26 ] and propagated by plate lysis of the host C. jejuni GM. C. jejuni GM cells were collected from a BA plate and suspended in 10 mM MgSO4 (Thermo Fisher) to a cell density of approximately 10⁷ CFU/ml. A volume of 500 μl of the cell suspension was added 10⁷ PFU DA10 before adding 5 ml of molten NZCYM top agar (0.6%, VWR Leicestershire, UK) and pouring on top of a NZCYM agar plate.

Jojoba oil was donated by Purcell Jojoba International (Avila Beach, CA); formic acid, 90.4% and hydrogen peroxide (50%), acetic anhydride, propionic and valeric anhydrides were purchased from Sigma-Aldrich (St. Louis MO); whereas MgSO₄, Na₂CO₃, NaCl, HCl, NaHCO₃, and p-TsOH were from Thermo Fisher (Chicago, IL). High-oleic sunflower oil, with 81% oleic acid, was obtained from Columbus Foods Company (Des Plaines, IL). Polyalpha olefin of viscosity 6 cSt at 100°C (PAO-6) supplied under the trade name Durasyn 166 by Ineos Oligomers (League City, TX) was a free sample. All reagents and solvents were used as supplied. Steel balls used in 4-ball experiments were obtained from Falex Corporation (Aurora, IL) and have the following specifications: material, chrome-steel alloy made from AISI E52100 standard steel; hardness, 64–66 R_c; diameter, 12.7 mm; and finish, grade 25 extra polish. The steel balls were cleaned prior to use in 4-ball experiments by consecutive 10 min sonications in isopropyl alcohol and hexane solvents.

E12.5 embryos were electroporated ex utero by injecting 100 ng/μl DNA in electroporation buffer (30 mM HEPES pH 7.5 [Thermo, #BP299-1], 300 mM KCl [Thermo, #BP366-1], 1 mM MgCl₂[Thermo, #BP214-500], and 0.1% Fast Green FCF [Thermo, #F99-10]) into the central canal of the neural tube. A BTX ECM 830 electroporator (BTX Harvard Apparatus, #45-0662) was used for bilateral electroporation into spinal cord neurons (five 30 V pulses, each of 50 ms duration for each half of the spinal cord). Following electroporation, dorsal spinal cords were dissected out and cut into explants for the explant outgrowth assay or used for preparation of dissociated neuronal cultures. For neuron culture, dissected spinal cords were washed in Hanks’ Balanced Salt Solution (HBSS, Gibco, #14175-079) and digested with 0.05% trypsin (Gibco, #25300054) for 7 min at 37°C. 1 μl of DNase I (NEB, #M0303L) and 0.15% MgSO₄ (Thermo, #7487-88-9) was added for an additional minute, and the samples were centrifuged at 400 × g for 4 min. Samples were washed with pre-warmed HBSS, and a small fire-polished Pasteur pipette was used to triturate the tissue and dissociate it into single cells. Cells were plated on acid-washed, poly-D-lysine and N-cadherin-coated coverslips and cultured in plating media (Neurobasal [Gibco, #21103-049] medium supplemented with 10% heat-inactivated FBS and 1X penicillin/streptomycin/glutamine).