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Briefly, 30 l of reaction mixture from each time point was heat-inactivated immediately at 80 C for 60s and allowed to cool on ice for 5 min

Adoptive transfer assay to induce EAU T cells isolated from the DLNs of EAU mice (day 14 after immunization) were stimulated using IRBP 1-20 (10 g/mL) under Th17-polarizing conditions, with or without melatonin (200 ng/mL) for 72 h, and were washed three times before administration to C57BL/6J mice (2 10 7 living cells/mouse) via intraperitoneal injection

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10.3390/ijms21113793 30