Pe conjugated anti c kit

For BrdU incorporation analysis in BM KSL cells in vivo, adult C57Bl/6 mice were irradiated with 750 cGy TBI and treated with 5 mg/kg DJ001 or vehicle daily from day + 1 to day + 10. On day + 10, mice were injected intraperitoneally with 2 mg BrdU (BD Biosciences). Sixteen hours later, BM cells were collected and stained with anti-BrdU FITC (BD Biosciences, #557891, 1:50), APC-Cy7-conjugated anti-Sca1 (BD Biosciences, #560654, 1:100), PE-conjugated anti-c-kit (BD Biosciences, #553355, 1:100), and V450 lineage cocktail (BD Biosciences, #561301, 1:10). We repeated this BrdU incorporation analysis on donor CD45.2⁺ cells at day + 7 and day + 21 following competitive transplantation into recipient CD45.1⁺ mice.

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).