Zaq fixable viability kit

Flow cytometry analysis routinely included the staining with ZAq Fixable Viability kit (BioLegend), which was carried out before the immunostaining for cell surface markers. Flow cytometry analysis of surface molecules or intracellular cytokines was performed in the viable population (ZAq^–). In some experiments, to gain a more detailed view of dying cells, the extent of apoptosis/necrosis was determined with a commercial kit (Pacific Blue Annexin V Apoptopsis Detection Kit with 7-AAD; 640926; BioLegend).

Naïve (CD3⁺ CD4⁺ CD44^– CD62L⁺) T cells were isolated from the spleen of Drd5^+/+ or Drd5^–/– mice by cell sorting using a FACS Aria II (BD), obtaining purities over 98%. Gut tropism was imprinted by activation of T cells in the presence of RA and IL-2 as described previously.³¹ (link) Briefly, naïve T cells were resuspended (10⁶ cells/mL) in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, MEM nonessential amino acids 1× and sodium pyruvate 1×, gentamicin 50 μg/mL, and β-mercaptoethanol 1 μg/mL (all from Gibco [Gaithersburg, MD], Thermo Fisher Scientific). Cells were activated with anti-CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a beads-to-cells ratio of 1:1 in the presence of 100 nM all-trans RA (Sigma-Aldrich) and 1000 U/mL recombinant mouse IL-2 (PeproTech, Rocky Hill, NJ) for 5 days. Viability and gut tropism were routinely confirmed after 5 days of culture by staining with ZAq Fixable Viability kit (BioLegend) and CCR9 and α4β7 immunostaining followed by flow cytometry analysis.