Pe cy7 conjugated anti fcεriα

Naïve Balb/c and ΔdblGata mice were euthanized and their peritoneal cavity was flushed by injecting 10mL of PBS in 10% FBS into peritoneal cavity, massaging the abdomen, and then drawing out the fluid. Cells were kept on ice, centrifuged at 500g for 5 mins, and supernatant removed. Pellets were lysed with 5mL Red Blood Cell Lysis Buffer (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes at room temperature, 5mL of RPMI 1640 media added to neutralize lysis, and centrifuged for 5 mins. Pellet was re-suspended in media and counted. Cells were stained with phycoerythrin (PE)- conjugated anti-c-Kit (BD Pharmingen, Mountain View, CA, USA), PE-cy7-conjugated anti-FcεRIα (Biolegend, San Diego, CA, USA) and biotinylated anti-T1/ST2. They were subsequently counterstained with Pacific Blue labelled streptavidin. Analysis was performed on FACS Canton II (BD Bioscience, Mountain View, CA, USA) and analyzed using Flowjo (Flowjo Software, Ahsland, OR, USA).

Lamina propria cells from SI of ΔdblGata or WT mice were stained with phycoerythrin (PE)- conjugated anti-c-Kit, fluorescein isothiocyanate (FITC)-conjugated anti-β7 integrin, Horizon V500-conjugated CD4, APC-Cy-7-conjugated anti-CD3e (BD Pharmingen, Mountain View, CA, USA), allophycocyanin (APC)-conjugated anti-IL-17RB, PE-Cy7-conjugated anti-FcεRIα (Biolegend, San Diego, CA, USA) and biotinylated anti-T1/ST2. Subsequently, cells were counterstained with PerCP-Cy5.5-conjugated monoclonal antibodies against lineage (Lin) markers (CD11b, CD11c, CD45R (BD Pharmingen, Mountain View, CA, USA) CD8α, Ly6G, and Pacific Blue labelled Streptavidin (Biolegend, San Diego, CA, USA)) before analysis with a FACS Canton II (BD Bioscience, Mountain View, CA, USA). CD4⁺ Th₂ cells were identified as Lin^-, CD3⁺, CD4⁺, and IL17RB⁺ populations. ILC2 cells were identified as Lin^-, CD3^-, CD4^-, 1L17RB⁺, and c-Kit^- populations. MMC9 cells were identified as Lin^-, CD3^-, CD4^-, 1L17RB^-, c-Kit⁺, and FcεRIα⁺ populations as previously described [38 (link)]. All cytometric data was acquired using BD FACSCanto II and data analysis was performed using Flowjo software (FlowJo, Ashland, OR, USA).

Femur and tibia were harvested from age and gender matched WT and ΔdblGata mice and bone marrow cells were cultured in mIL-3 and mSCF (20ng/mL each) as previously described [39 (link)]. Mast cell maturation and purity was tracked weekly via FACS analysis for seven weeks. Cells were stained with phycoerythrin (PE)- conjugated anti-c-Kit (BD Pharmingen, Mountain View, CA, USA), PE-cy7-conjugated anti-FcεRIα (Biolegend, San Diego, CA, USA) and biotinylated anti-T1/ST2. Analysis was performed on FACS Canton II (BD Bioscience, Mountain View, CA, USA).