Magnetic bead isolation

c-Kit+ CPCs were isolated from 8 week old male wild-type and Abi3bp knockout litter-mates. Minced ventricular tissue was digested in 100U of collagenase in Hank's Buffered Saline solution at 37° C for 15 minutes. Single cells were passed through a 100 μm sieve and low density cells were separated on a discontinuous Percoll gradient. Primary cells were cultured for 3 days in CPC-maintenance media (DMEM/F12-K 1:1, 20% ES cell qualified FBS, 10 ng/mL bFGF, 20 ng/mL EGF, 100U LIF, and 1x ITS (insulin-transferrin-selenium)). c-Kit⁺ cells were then selected by magnetic bead isolation (Miltenyi Biotech, Boston MA) and further cultured in CPC-maintenance media. Cells were differentiated at passage 3. At this passage the cells were positive for c-Kit and CD29 (Online Figure IA). Apoptosis and necrosis was not significantly different between c-Kit+ CPCs derived from wild-type and Abi3bp knockout mice (Online Figure IB). In CPC-maintenance media, Abi3bp knockout c-Kit+ CPCs expressed significantly lower levels of Abi3bp, Mef2C, and cardiac troponin-I (cTroponin-I) when compared to wild-type c-Kit+ CPCs, however, expression of Gata4 and Gata6 was not significantly different between wild-type and Abi3p knockout c-Kit+ CPCs (Online Figure IC). CPCs were not observed to beat during the experiments.

MLR were performed, as we described, with minor modifications (31 (link), 34 (link)). Responder T-lymphocytes were initially enriched using magnetic activated cell sorting, labeled with 5 μM CFSE for 10 minutes at 37 °C, and plated at 1×10⁴ cells/well. Brain tissue was harvested at 24h post-sham/TBI and CD11b⁺ CD68⁺ F4/80⁺ brain macrophages were consecutively sorted three times by magnetic bead isolation (Miltenyi Biotech). Purified brain macrophages were used as stimulators following plating at 5×10⁴ cells/well. Combinations of responders (naïve T-lymphocytes) and stimulators (brain macrophages) were prepared in triplicate wells. Cells were cultured in 200 μL/well of RPMI 1640 medium supplemented with fetal bovine serum, penicillin, streptomycin, L-glutamine, and 2-mercaptoethanol. After 72 – 96h of incubation at 37°C in a humidified, 5% CO₂ incubator, cells were harvested into flow cytometry tubes. Following a PBS wash, samples were incubated at 4°C in for 20 minutes in the dark with anti-rat CD71-phycoerythrin-conjugated antibody to label activated and dividing T cells. Samples were then washed with PBS and T-cell proliferation and polarization phenotype was quantified in triplicate by flow cytometry.

Donor cells were prepared by isolating naïve CD4⁺ T cells (CD4⁺CD25⁻CD45RB^hi) by magnetic bead isolation (Miltenyi) from WT and CD4^ΔMthfd2 littermates aged 8–12 weeks. 400,000 naïve CD4⁺ T cells were i.p. injected into recipient male Rag1^−/− mice aged 8 weeks to induce colitis. Mice were weighed twice weekly to monitor disease progression. Mice were euthanized after 7 weeks and spleen and mesenteric lymph nodes were collected for T cell phenotyping.