Efluor450 cocktail

Single cell suspensions were prepared as previously described 15 (link), and contaminating erythrocytes were lysed using ammonium chloride buffer where appropriate. Cell suspensions were stained with optimal dilutions of antibodies directly conjugated to either Biotin, FITC, PE, PE-Texas Red, PE-Cy5.5, PerCP-Cy5.5, PerCP-eFluor710, PE-Cy7, Pacific Blue, eFluor 450, allophycocyanin, Alexa Fluor 647, or allophycocyanin-eFluor 780. Biotinylated antibodies were visualized using streptavidin PE-Texas Red (BD Bioscience). Lineage positive cells were gated out of whole BM suspensions using mouse hematopoietic lineage eFluor450 cocktail (eBioscience).The following specific antibodies were used to detect cell surface antigens: CD117/c-Kit (2B8), Sca-1/Ly6A/E (D7), CD127/IL-7Rα (A7R34), CD34 (RAM34), CD135/Flt3 (A2F10), CD25 (PC61.5), AA4.1 (AA4.1), CD27 (LG.7F9), CD150 (mShad150), CD48 (HM48-1), CD244.2 (eBio244F4), CD45.1 (A20), CD45.2 (104), GR-1/Ly-6G (RB6-8C5), CD11b (M1/70), CD11c (N418), CD45R/B220 (RA3-6B2), CD19 (eBio1D3), CD3 (145-2C11), CD4 (GK1.5), and CD8 (53-6.7). All antibodies were obtained from eBioscience. The CD1d PBS-57 tetramer was obtained from the NIH Tetramer Core Facility (Emory University, Atlanta, USA).

Progenitors in the peripheral blood were identified and quantified using a protocol adapted from Serwold et al. 40 (link). Peripheral blood from six animals per group was collected in EDTA by terminal cardiac bleed and pooled. Blood was diluted 1:1 in RPMI and mononuclear cells enriched by centrifuging through a layer of Lympholyte-Mammal (Cedarlane Laboratories, Burlington, USA). Cells were collected, washed, and stained for Lin markers (hematopoietic lineage eFluor450 cocktail (EBioscience) and NK1.1, CD11b, and CD19 on FITC), CD27-PerCP-eFluor710, CD127-Alexa Fluor 647, Sca-1-Pe-Cy7, CD117-allophycocyanin-eFluor780, and Flt3-PE (all eBioscience). At least 5 × 10⁶ events were analyzed by flow cytometry per sample.