Human whole leukocytes from anonymous healthy donors were isolated from apheresis cones from ARUP using RBC sedimentation with HetaSep (STEMCELL Technologies, Vancouver, British Columbia). Freshly isolated whole leukocytes were used fresh for in vitro culture.
Whole leukocytes were cultured with or without 5-A-RU (50 nM) and MGO (50 µM) in 200 µL RPMI complete media and incubated at 37 °C overnight. CD69, and eotaxin, granzyme A, and in MAIT cells and Eosinophils were analyzed using flow cytometry. To detect released cytokines in the medium, 25 µL of supernatant was analyzed using Cytokine 35-Plex Human Panel for the Luminex™ platform (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on the Luminex MAGPIX. To block interactions activation of MAIT cell via MR1, anti-MR1 (Clone 26.5, Biolegend, San Diego, CA, USA) or an isotype control was used at 10 µg/ml.
Whole leukocytes were cultured with or without 5-A-RU (50 nM) and MGO (50 µM) in 200 µL RPMI complete media and incubated at 37 °C overnight. CD69, and eotaxin, granzyme A, and in MAIT cells and Eosinophils were analyzed using flow cytometry. To detect released cytokines in the medium, 25 µL of supernatant was analyzed using Cytokine 35-Plex Human Panel for the Luminex™ platform (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on the Luminex MAGPIX. To block interactions activation of MAIT cell via MR1, anti-MR1 (Clone 26.5, Biolegend, San Diego, CA, USA) or an isotype control was used at 10 µg/ml.