Wt and Pla2g5-null mice received four doses of 25ug of Alternaria in 20ul of PBS i.n. on day 0, 3, 6 and 9 and euthanized 18h later in order to expand ILC2s prior to FACs sorting. Sorting of ILC2s (CD45+ Lin− (CD3, CD19, Ly6g, CD11c, CD11b, Nk1.1, FcεR1−), Thy1.2+) was performed using a FACSDiva 8.0.1 cell sorter (BD Bioscience). Purified CD45+ lin-Thy1.2+ cells (>98%) were rested for 40h with 10ng/mL rIL-2 and rIL-7 (R&D Systems, Minneapolis, MN) in 96 well around bottom plates (20000 cells per well). Prior to stimulation, the medium was changed to fresh medium. ILC2s were cultured with 30ng/mL rIL-33 (R&D Systems), 200 μM LinOleic Acid (Cayman Chemical) or 200 μM Oleic Acid (Cayman Chemical)22 (link) or all together for 8h. For intracellular cytokine staining, 1 μl/mL of Golgi Plug (BD Bioscience) was added to ILC2s 6h before collection for FACs analysis.
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Expansion and Stimulation of ILC2s from Murine Models
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Other organizations : Brigham and Women's Hospital, Harvard University, University of California, San Diego
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Variable analysis
independent variables
- Genotype (Wt and Pla2g5-null mice)
- Alternaria treatment (25ug in 20ul of PBS i.n. on day 0, 3, 6 and 9)
- Expansion of ILC2s prior to FACS sorting
- Cytokine production by ILC2s after 8h of stimulation with IL-33, Linoleic Acid, Oleic Acid, or all together
- Cell sorting procedure (CD45+ Lin- Thy1.2+ cells >98% purity)
- Resting of ILC2s for 40h with 10ng/mL rIL-2 and rIL-7 prior to stimulation
- Golgi Plug addition 6h before collection for FACS analysis
- Positive control: Stimulation with all three treatments (IL-33, Linoleic Acid, Oleic Acid) together
- Negative control: Not explicitly mentioned
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