Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1(-/-) Mice

Zhang, Yongli; Zhang, Hangjie; Ma, Wenqiang; Liu, Kefang; Zhao, Min; Zhao, Yingze; Lu, Xuancheng; Zhang, Fuping; Li, Xiangdong; Gao, George F.; Liu, William J.

. Remove the supernatant. 14. Resuspend the cells with 10 mL of ice-cold RPMI/FBS medium and centrifuge them at 600 x g for 5 min at 4 °C. Remove the supernatant. 15 . Resuspend the cells with 10 mL of complete RPMI medium and count the number of cells as in step 3.9. mM NaCl] to the chamber and concentrate it to a final volume of 30-50 mL. 4. Transfer the refolding solution to a centrifuge tube and spin it at 2,500 x g for 15 min at 4 °C. 5. Carefully transfer the supernatant to a 10 kDa centrifugal filter and further concentrate it to a final volume of 500 µL at 2,500 x g for 30 min. 6. Transfer the supernatant to a fresh tube and spin it at 12,000 x g for 15 min. Purify the protein with S200 10/300 GL gel filtration chromatography. 7. Collect the MHC complex peak and concentrate it to a final volume of 350 µL. 8. Apply the multimerized reagents into a 100 kDa spin tube and concentrate it by centrifugation at 2,000 x g at 4 °C to a volume of <100 µL. 9. Dilute the sample in the spin tube to 4 mL using PBS (pH 8.0) and concentrate it again to <100 µL. 10 . Repeat the buffer exchange step in PBS (pH 8.0) 4x. 11. Fill up the total volume again to 500 µL using PBS (pH 8.0). Concentrate the sample to an estimated concentration of 2-2.5 mg/mL at 2,000 x g at 4 °C. Store the sample in the dark at 4 °C.