Anti cd19 apc cy7 1d3

Bone marrow cells were collected from left femurs and tibias of 5 female eKO and 5 female WT mice by removing both epiphyses and flushing out cells with PBS containing 3% FBS. The cells were then washed and incubated with anti-mouse CD16/CD32 (mouse BD Fc-block, catalog number 553141, BD Biosciences, San Jose, CA) for 5 minutes on ice. Then the samples were stained for 30 minutes on ice with the following antibodies: 5 μg/ml anti-CD3-FITC (145-2C11, BD Biosciences) for T cells, 2 μg/ml anti-CD19-APC-Cy7 (1D3, BD Biosciences) for B cells, 0.5 μg/ml anti-CD11b/Mac1-APC (M1/70, BD Biosciences) for monocyte progenitors, and 0.5 μg/ml anti-Ter119-PerCP/Cy5.5 (Ter-119, BD Biosciences) for erythroid cells. Cells were washed to remove unbound antibodies and then fixed with 4% paraformaldehyde in phosphate-buffered saline for 20 minutes before analysis using a BD FACS Aria flow cytometer (BD Biosciences). Data analysis was conducted using FlowJo Software (FlowJo, LLC, Ahsland, OR). Fluorescence Minus One (FMO) controls (BD Biosciences) guidance was followed for appropriate gating for the different cell populations.

Bone marrow cells were collected by removing both ends of femurs and flushing out the cells with PBS containing 3% FBS. Single-cell suspensions of spleen cells were prepared by grinding the spleen through a cell strainer using a syringe plunger. Bone marrow and spleen cells were then washed and incubated with anti-mouse CD16/CD32 (mouse BD Fc-block, catalog number 553141, BD Biosciences, San Jose, CA) for 5 min. The cells were stained for 30 min using the following antibodies: 2 µg ml⁻¹ anti-CD19-APC-Cy7 (1D3, BD Biosciences) to identify B cells and 5 µg ml⁻¹ anti-CD3-FITC (145-2C11, BD Biosciences) to identify T cells. After the cells were washed to remove unbound antibodies, the samples were analyzed by a BD FACS Aria flow cytometer (BD Biosciences). An example of the gating strategy is presented in Supplementary Fig. 4. The data were then analyzed using FlowJo Software (FlowJo, LLC, Ashland, OR). The guidance of Fluorescence Minus One (FMO) controls (BD Biosciences) was followed to draw appropriate gates for the cell populations.