Edc nhs

PG2 was prepared through a combined salt-leaching/crosslinking method. First, 1 g of sodium chloride (NaCl) was added into a 3.5 cm petri dish as the porogen, followed by the mixture of 100 μL 0.2 g mL⁻¹ pullulan (Sigma-Aldrich, Oakville, ON, Canada) aqueous solution and 200 μL 0.1 g mL⁻¹ gelatin (Type A, BioShop, Burlington, ON, Canada) aqueous solution, and an extensive mixing action. Second, 10 mg trisodium trimetaphosphate (STMP) (Sigma-Aldrich) with 5 μL 10N sodium hydroxide solution (NaOH) was mixed in 200 μL water and then added into the petri dish, followed by an extensive mixing action. The final mixture in the petri dish was then put into an oven at 60 °C for 3 h. The solidified resultant was washed by PBS at 4 °C until pH on the material was maintained at pH 7. In order to crosslink gelatin, 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / 80 mM N-hydroxysuccinimide (EDC/NHS, Thermo Fisher Scientific, Waltham, MA, USA) aqueous solution was prepared right before use, and 2 mL of EDC/NHS solution was added onto the solidified material for crosslinking. The reaction was conducted at 4 °C overnight, and the reactant was washed with PBS for 12 h by changing the medium every 2 h. The final product was freeze-dried and stored at 4 °C before use.

For immunization, recombinant drTLR5_N14 was covalently conjugated to fixed Jurkat T cells as a carrier to enhance the immune response. Jurkat T cells were cultured in RPMI-1640 (Cellgro) containing 10% fetal bovine serum (Hyclone) at 37 °C and 5% CO₂. For conjugation, 10⁸ Jurkat T cells were washed three times with PBS, then fixed overnight in 4% paraformaldehyde (Electron Microscopy Sciences). The following day, the cells were washed in 20 mM MES, pH 5.5, and activated for amine conjugation by treatment with (1-ethyl-3-(–3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS, ThermoFisher) for 20 min at room temperature, then washed briefly with PBS. 0.5 mg/mL drTLR5_N14 in PBS was immediately added to the EDC/NHS-activated cells and incubated for 3 hrs at room temperature on a rotating mixer. After the incubation, the cells were washed with PBS/10 mM Tris, pH 7.5, and the drTLR5-conjugated Jurkat cells were stored at 4 °C until needed for immunization.