Cd11b mac 1 clone m1 70

A whole thymus was gently washed and strained through a 70 μm mesh to prepare single cell suspensions. 10 × 10⁶ thymic cells were stained in 200 μl PBS supplemented with 2% FCS containing the following antibody solution: PE-conjugated lineage cocktail with antibodies against CD3e (clone 145-2C11, eBioscience), Ly-6G/Gr-1 (clone RB6-8C5, eBioscience), CD11b/Mac-1 (clone M1/70, BD PharMingen), CD45R/ B220 (clone RA3-6B2, BD PharMingen) and TER-119 (clone Ter119, BD PharMingen); CD4 (BV421, clone H129.19, BD PharMingen), CD8a (APC, clone 53-6.7, BD PharMingen), CD44 (PerCP-Cy5.5, clone IM7, eBioscience), CD25 (PE-Cy7, clone PC61.5, eBioscience)

Bone marrow cells were isolated from femurs, tibiae and iliac crests with PBS supplemented with 2% FCS and strained through 70 μm meshes. Red cells were lysed by resuspending the cells in 10 mL red cell lysis buffer (130-094-183, MACS Miltenyi Biotec) for 10 min at room temperature. After centrifugation, the cell pellet was resuspended in PBS supplemented with 2% FCS and nucleated cells were counted with 3% acetic acid on a Vi-Cell XR cell viability counter (Beckman Coulter). 10 × 10⁶ bone marrow cells were resuspended in 200 μL of PBS supplemented with 2% FCS containing the following antibody solution: FITC-conjugated lineage cocktail with antibodies against CD4 (clone H129.19, BD PharMingen), CD3e (clone 145-2C11, eBioscience), Ly-6G/Gr-1 (clone RB6-8C5, eBioscience), CD11b/Mac-1 (clone M1/70, BD PharMingen), CD45R/ B220 (clone RA3-6B2, BD PharMingen), FcεR1α (clone MAR-1, eBioscience), CD8a (clone 53-6.7, BD PharMingen), CD11c (clone N418, eBioscience), TER-119 (clone Ter119, BD PharMingen); c-Kit (PerCP-Cy5.5, clone 2B8, eBioscience), Sca-1 (PE-Cy7, clone D7, eBioscience), Flt3 (PE, clone A2F10, eBioscience), CD34 (eFluor660, clone RAM34, eBioscience), CD16/32 (BV421, clone 93, BioLegend) and Il-7R⍺ (BV605, clone A7R34, BioLegend).