Fortessa instrument

Noncompetitive transplants were performed with 1 million BM cells from 6–8-wk-old Ikzf2^fl/fl vavcre- or Ikzf2^fl/fl vavcre+ mice, injected into lethally irradiated B6SJL congenic CD45.1 recipients. In the primary transplants, chimerism was checked from 8 to 34 weeks, every 4 weeks by either assessing chimerism via peripheral blood or bone marrow aspirates. Mice were sacrificed 34 weeks after injection for primary transplant and 24 weeks for secondary transplant experiments. For chimerism, either peripheral blood or bone marrow cells was extracted and subjected to red blood cell lysis. For chimerism assessment in mature cells of the different lineages, Abs for following markers were used: Mac1, Gr1, c-Kit, CD71, Ter119, B220, CD19, IgM, CD3, CD4, CD8, CD45.2 and CD45.1. For chimerism assessment in stem cells and progenitor cells, following cocktail panel was used: (Lineage; CD3, CD4, CD8, Gr1, B220, CD19, and TER119 all conjugated with PeCy5), Sca-Pac Blue, CD34-FITC, SLAM-APC, CD48-PE, c-KIT-APC-Cy7, FcgRIIb-PeCy7, CD45.2-Alexa700 and CD45.1-PE Texas-red. Stained cells were then analyzed on the BD Fortessa instrument. Following markers were used to define different cell population : HPCs (Lin⁻c-Kit⁺Sca1⁻), GMPs (LK, FcgRIIb^HighCD34⁺), CMPs (LK, FcgRIIb^Mid CD34⁺), MEPs (LK, FcgRIIb^Low CD34⁻), B cells (B220^+, CD19, IgM), T cells (CD3⁺, CD4, CD8) and erythroid (Ter119, CD71).

Preparations of spleen and MLN suspensions were performed as described previously (20 (link)). Intestinal lamina propria cells were isolated from either cecal or colonic tissue by EDTA incubation, collagenase/DNAaseI digestion and Percoll gradient (see (20 (link)) and Supplementary Methods). For analysis of cellular cytokine production, 10^6 cells were incubated per well of a 96 well plate at 37C for 4h in supplemented RPMI (3% FBS) containing phorbol myristate acetate/ionomycin and Brelfeldin A. Unstimulated controls were included with Brelfeldin A alone.
For flow cytometric analysis cells were incubated with cell surface antibodies (see below) and AQUA Live/Dead stain (Invitrogen, San Diego, CA) prior to fixation, permeabilization and intracellular staining with buffers from the FoxP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA). Cell surface antibodies used were CD3, CD4 and TCRβ and intracellular antibodies used were IL-17, IFNγ (cytokine panel) and FoxP3 and Helios (Treg panel). For antibody clones, conjugates and suppliers see Supp. Table 3. All flow cytometry samples were acquired with a BD Fortessa instrument (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR). Frequencies of cytokine-producing cells represent the stimulated sample value minus that of its unstimulated control.