Lamina propria mononuclear cells (LPMC) were isolated from colonic tissue as previously described20 . LIVE/DEAD fixable aqua dead cell stain kit (Molecular Probes) was used to select live cells. For analysis related to transcription factors, large intestine LPMC cells were stained with anti-CD3- E780 (eBiosciences 17A2) and anti-CD4-AF700 (eBiosciences GK1.5) before fixation and permeabilization with Cytoperm/Cytofix flowed by intracellular staining against anti-Foxp3-E450 (eBiosciences FJK-16s) and anti-RORγt-PE (eBiosciences B2D). For analysis related to cytokine, LPMC cells were stimulated with phorbol myristate acetate (PMA) and ionomycin with BD GolgiPlug for 3.5 hours. Following surface-marker staining with anti-CD3- E780 (eBiosciences 17A2) and anti-CD4-AF700 (eBiosciences GK1.5), LPMC cells were prepared as per manufacturer’s instruction with Cytoperm/Cytofix (BD Biosciences) for intracellular cytokine evaluation of IL-17A (eBiosciences 17B7) and IFNγ (eBiosciences XMG1.2).
Lima S., Gogokhia L., Viladomiu M., Chou L., Putzel G., Jin W., Pires S., Guo C.J., Gerardin Y., Crawford C.V., Jacob V., Scherl E., Brown S.E., Hambor J, & Longman R. (2021). Transferable IgA-coated Odoribacter splanchnicus in Responders to Fecal Microbiota Transplantation for Ulcerative Colitis Limits Colonic Inflammation. Gastroenterology, 162(1), 166-178.
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