Pe cy7 conjugated sca 1

Monoclonal antibodies against CD16/CD32, Alexa Fluor 700-conjugated CD45.2, fluorescein isothiocyanate-conjugated lineage cocktail (CD3ε, Gr-1, CD11b, CD45R, and Ter119), CD19, and CD49b, phycoerythrin (PE)-conjugated CD127 and IL-5, peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated CD25, allophycocyanin-conjugated c-Kit and IL-4, PE-Cy7-conjugated Sca-1 were purchased from BioLegend. Streptavidin-conjugated brilliant violet (BV) 421, carboxyfluorescein diacetate succinimidyl ester (CFSE), zombie NIR dye, and brefeldin-A were also purchased from BioLegend. Monoclonal antibodies against biotinylated T1/ST2 and PerCP-eFluor710-conjugated IL-13 were purchased from MD Bioproducts and eBioscience, respectively. Propidium iodide (PI) was purchased from BD Biosciences. Murine IL-2 and IL-33 were purchased from PeproTech.

For isolating HSPCs from bone marrow, magnetic‐activated cell sorting (MACS), and fluorescence‐activated cell‐sorting (FACS) analysis were performed as follow. Bone marrow cells were harvested from donor femur and tibia by crushing bones with mortar and pestle in PBS containing 2% FCS and filtering through a 40 μm mesh filter. After red blood cells were lysed, bone marrow cells were incubated with a cocktail of biotin‐conjugated lineage‐specific antibodies against CD45, Gr1, CD11b, Ter119, CD4, CD8, CD3 (553,029, 553,060, 553,086, 553,125, 553,309, 553,672, and 553,728, respectively, BD Biosciences), and anti‐Biotin microbeads for MACS (130–090‐485, Miltenyi Biotec). The separation of lineage‐negative cells was performed with an autoMACS pro separator (Miltenyi Biotec). Lineage‐depleted cells stained with APC‐conjugated c‐Kit (17–1171‐81, eBioscience) and PECy7‐conjugated Sca‐1 (108,116, Biolegend) antibodies were sorted using FACS Aria II (BD Biosciences) with over 95% purity. The DsRed fluorescence expression in the purified HSPCs was over 99% by FACS DiVa software (BD Biosciences).