Gut Tissue Lymphocyte Isolation and EliSPOT

Gut tissues were prepared as previously described (15 (link)). Briefly, cleaned gut tissues were deepithelialized in 5 mM ethylenediaminetetraacetic acid (EDTA), 15 mM Hepes, and 5% fetal bovine serum (FBS) in Hank’s Balanced Salt Solution (HBSS) before digestion with 0.5 mg/mL Collagenase Type IV (Sigma Aldrich) and 0.25 mg/mL DNase 1 in HBSS with 2% FBS. Lymphocytes enriched by passing the cell suspension sequentially through 100- and 40-μm strainers. For enzyme-linked immune absorbent spot (EliSPOT) experiments, CD4⁺ T cells were isolated from mLN using magnetic isolation (CD4 microbeads, Miltentyi Biotech), and dendritic cells were isolated from the spleens of naive SPF C57BL/6 mice (Jackson Laboratories) by magnetic isolation (CD11c microbeads, Miltenyi Biotech) using an AutoMACS instrument (Miltenyi Biotech) following the manufacturer’s instructions.

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Britton G.J., Contijoch E.J., Spindler M.P., Aggarwala V., Dogan B., Bongers G., San Mateo L., Baltus A., Das A., Gevers D., Borody T.J., Kaakoush N.O., Kamm M.A., Mitchell H., Paramsothy S., Clemente J.C., Colombel J.F., Simpson K.W., Dubinsky M.C., Grinspan A, & Faith J.J. (2020). Defined microbiota transplant restores Th17/RORγt+ regulatory T cell balance in mice colonized with inflammatory bowel disease microbiotas. Proceedings of the National Academy of Sciences of the United States of America, 117(35), 21536-21545.

Publication 2020

Collagenase Type IV SPF C57BL/6 mice CD11c microbeads AutoMACS instrument

C57bl mice Cd4 t cells Dendritic cells Digestion Dnase Elispot Enzyme Ethylenediaminetetraacetic acid Fetal bovine serum Hepes Isolation Lymphocytes Microbeads Salt Tissues Type iv collagenase

Corresponding Organization :

Other organizations : Icahn School of Medicine at Mount Sinai, Cornell University, Janssen (United States), Centre For Digestive Diseases, UNSW Sydney, St Vincent's Hospital, University of Melbourne, Concord Repatriation General Hospital, University of Sydney, Macquarie University

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Variable analysis

independent variables

Collagenase Type IV (0.5 mg/mL)
DNase 1 (0.25 mg/mL)

dependent variables

Lymphocyte enrichment by passing the cell suspension through 100- and 40-μm strainers
CD4+ T cell isolation from mesenteric lymph nodes (mLN)
Dendritic cell isolation from spleens of naive SPF C57BL/6 mice

control variables

Cleaned gut tissues
5 mM ethylenediaminetetraacetic acid (EDTA)
15 mM Hepes
5% fetal bovine serum (FBS) in Hank's Balanced Salt Solution (HBSS)
HBSS with 2% FBS
Naive SPF C57BL/6 mice

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