V450 conjugated anti cd45

Spleen single-cell suspensions were collected from the mice, filtered with a cell strainer, distributed into 2 sets and were suspended in 50 μL of cold PBS containing 2% fetal calf serum. The first set of cells was stained with V450-conjugated anti-CD45 (BD Bioscience), BB515-conjugated anti-major histocompatibility complex II (MHCII) (BD Bioscience), phycoerythrin-conjugated anti-CD11c (BD Bioscience) for the detection of activated DCs, anti-CD11b⁺/F4/80⁺ for macrophages, and anti-CD11b⁺/Ly6G⁺ for neutrophils. The other set of cells was stained with peridinin chlorophyll protein complex-conjugated anti-CD3 (BD Bioscience), fluorescein isothiocyanate-conjugated anti-CD4 (eBiosience, San Diego, CA, USA), APC-conjugated anti-CD8, and phycoerythrin-conjugated anti-Foxp3 for the detection of T cells. The cells were analyzed by flow cytometry (LSR II; BD Bioscience). Flow cytometry was performed using an BD FACSCantoIIand analyzed by FlowJo software.

Harvested BM cells from the femur and tibia were resuspended in PBS containing 2% FBS, incubated at 4°C for 30–40 min. with the following antibodies: V450-conjugated anti-CD45 (BD Bioscience), APC-conjugated anti-CD11b (BD Bioscience), V450-conjugated anti-CD3e (BD Bioscience) and APC-conjugated anti-B220 (BD Bioscience). After the antibody incubation, cells were washed, centrifuged at 1500 rpm for 5 min. and resuspended in PBS containing 2% FBS. Analysis and cell sorting were performed by FACS AriaII (BD Bioscience). Cell purity was verified as more than 95%. Data were analyzed with the FlowJo software (FlowJo, LLC).