Lsrfortessa 1

The cells were incubated with the antibodies against cell surface antigens after blocking with anti-CD16/CD32. The cells were then fixed with paraformaldehyde (2%), permeabilized with saponin (0.5%), and incubated with antibodies against intracellular antigens if needed. For interferon-gamma (IFNγ) and granzyme B staining, cells were treated with brefeldin A (BFA, 3 μg/ml) and monensin (2 μM) for 4 h before they were stained for FACS analysis. Data were acquired and analyzed by Accuri C6 or BD LSRFortessa I (BD Biosciences, Bedford, MA, USA) and the FlowJo software (24 (link), 25 (link)).

Peritoneal wash, lymph node (LN) and MC analyses were performed as previously described (42 (link)). In brief, mice were euthanized and a hypodermic needle was inserted into the abdominal cavity (18 gauge). Sterile saline + 10% Fetal Bovine Serum (FBS) (10 ml) was injected into the peritoneal cavity, the abdominal region was gently massaged for 1 min and the fluid removed. The peritoneal lavage was centrifuged at 1200 rpm for 5 min at room temperature. The supernatant was poured off and Red Cell Lysis Buffer (RCLB) (Sigma-Aldrich, St.Louis, MO, USA) was added. After RCLB, cells were counted and re-suspended in FACS buffer (PBS/ 0.5% BSA or 1% FCS). The single-cell suspensions were washed with FACS buffer (PBS/ 1% BSA or FCS) and incubated with combinations of the following Abs: CD16/CD32 combinations of the mAb-fluorochrome conjugates (100 μl PBS/BSA, 1 μl FcεRI-APC, 1 μl ST2-PerCP-Cy5.5, 0.5 μl cKit-PE-Cy7, 1μl CD19-APC, 1μl B220-PerCP-Cy5.5 or CD5-PE) at 4°C for 30 min in the dark. The cells were measured on a BD LSR Fortessa I or FACS Canto following the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA) and analyzed using the FlowJo 10 software (FlowJo LLC, Ashland, OR, USA). Data were expressed as mean fluorescence intensities (MFI) of labeled cells.